Compositions, kits, and methods for the diagnosis, prognosis, monitoring, treatment and modulation of post-transplant lymphoproliferative disorders and hypoxia associated angiogenesis disorders using galectin-1

ABSTRACT

The present invention is based, in part, on the discovery that galectin-1 (Gal1) plays a role in viral-associated PTLD, e.g., EBV-associated PTLD and hypoxia associated angiogenesis disorders. Accordingly, the invention relates to compositions, kits, and methods for diagnosing, prognosing, monitoring, treating and modulating viral-associated PTLD, e.g., EBV-associated PTLD and hypoxia associated angiogenesis disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage Application of InternationalApplication No. PCT/US2010/056547, filed on Nov. 12, 2010, which claimsthe benefit of priority to U.S. Provisional Application No. 61/335,779,filed on Jan. 12, 2010, U.S. Provisional Application No. 61/283,159,filed on Nov. 30, 2009, and U.S. Provisional Application No. 61/261,125,filed on Nov. 13, 2009; the entire contents of each of which applicationare expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Post-transplant lymphoproliferative disorders (PTLD) are potentiallyfatal conditions associated with immunocompromised solid organ and stemcell transplantation that can have 70-80% mortality (Gottschalk et al.(2005) Annu. Rev. Med. 56, 29-44; Paya et al. (1999) Transplantation 68,1517-1525). PTLD is often associated with viral infection, such thatlatent viral infection of the transplanted material can causecomplications in the transplant subject. For example, Epstein-Barr virus(EBV)-associated PTLD derives from herpes virus exposure thatestablishes latent infection in a majority of healthy adults.Proliferation of EBV-infected B cells in PTLD is maintained byexpression of EBV latent genes, such as latent membrane protein 1 (LMP1)and LMP2A, viral immune evasion strategies, and impaired host immunesurveillance. The incidence of PTLD varies according to the organtransplanted, as well as the intensity and duration ofimmunosuppression. In renal transplant recipients PTLD occurs in 1-2% ofpatients, but the incidence is as high as 20% in small bowel transplantand 1%-10% in lung, heart, liver, and kidney transplant recipients(Gottschalk et al. (2005) Annu. Rev. Med. 56, 29-44; Paya et al. (1999)Transplantation 68, 1517-1525). Children and transplant recipientswithout previously established anti-EBV immunity are among those atgreatest risk for development of a PTLD. There is no accepted standardof therapy for PTLD, and the progression of the disease in patients isoften not responsive to currently available therapies. Management ofearly PTLD lesions is currently based on reduction or withdrawal ofimmunosuppression which increases the risk of graft rejection.

In addition, cancer cells adapt to low oxygen tension by promoting theexpression of genes associated with anaerobic metabolism, invasion andangiogenesis (Pugh et al. (2003) Nat Med 9, 677-684; Fraisl et al.(2009) Dev Cell 16, 167-179). The concerted action of hypoxia-regulatedpathways allows tumor cells to sprout new vessels, co-opt host vesselsand/or recruit angio-competent bone marrow-derived cells to generatefunctionally abnormal tumor vasculatures (Ferrara et al. (2005) Nature438, 967-974). In spite of the well-established roles ofhypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor(VEGF), increasing evidence suggests the contribution of alternative‘non-canonical’ pathways to hypoxia-driven neovascularization (Ferrara,N. (2010) Cytokine Growth Factor Rev 21, 21-26). This proposition isfirmly grounded on emerging preclinical and clinical data demonstrating‘evasive resistance’ or ‘intrinsic refractoriness’ to VEGF-targetedtherapies, which fail to produce enduring clinical benefits (Ferrara, N.(2010) Cytokine Growth Factor Rev 21, 21-26; Ebos et al. (2009) CancerCell 15, 232-239; Paez-Ribes et al. (2009) Cancer Cell 15, 220-231).

The mechanisms underlying ‘evasive resistance’ involve revascularizationas a result of the delivery of alternative pro-angiogenic signals(Bergers et al. (2008) Nat Rev Cancer 8, 592-603) and/or mobilization ofbone marrow-derived inflammatory cells, which together with endothelialand pericyte progenitors, are recruited to the tumor vasculature(Shojaei et al. (2007) Nat Biotechnol 25, 911-920; Bergers et al. (2008)Nat Rev Cancer 8, 592-603). Future anti-angiogenic therapies mightcapitalize on an improved understanding of these compensatory pathways,as well as the elucidation of the molecular underpinnings of bloodvessel normalization and the identification of hallmark signatures whichdistinguish healthy from tumor-associated endothelium (Jain, R. K.(2005) Science 307, 58-62). Although substantial changes in theendothelial cell (EC) surface ‘glycome’ were apparent under differentculture conditions (Garcia-Vallejo et al. (2006) J Cell Physiol 206,203-210; Willhauck-Fleckenstein et al. (2010) Angiogenesis 13, 25-42),suggesting a role for glycan structures in differentially regulatingangiogenesis in hypoxic versus normoxic and in neoplastic versus healthytissues, the specific glycan structures, mediating molecules, andmechanisms were not known prior to the results described herein.

Programmed remodeling of cell surface glycans can control cellularprocesses by displaying or masking ligands for endogenous lectins(Paulson et al. (2006) Nat Chem Biol 2, 238-248; van Kooyk et al. (2008)Nat Immunol 9, 593-601). Recent efforts involving genetic manipulationof N- and O-glycosylation pathways have revealed essential roles formultivalent lectin-glycan lattices in the control of receptor signaling(Ohtsubo, et al. (2006) Cell 126, 855-867; Dennis et al. (2009) Cell139, 1229-1241; Dam et al. (2010) Glycobiology 20, 1061-1064). Regulatedglycosylation can control sprouting angiogenesis by modulating bindingof Notch receptor to its ligands Delta-like 4 (Dll4) or Jagged1(Benedito et al. (2009) Cell 137, 1124-1135), fine-tuning neuropilin-1(NRP-1) signaling (Shintani et al. (2006) EMBO J 25, 3045-3055) andfacilitating CD31-mediated homophylic interactions (Kitazume et al.(2010) J Biol Chem 285, 6515-6521). Yet, whether differentialglycosylation enables the formation of discrete lectin-glycan latticesand signaling clusters that are functionally relevant to angiogenesisremains largely unexplored.

In view of the above, it is clear that there remains a need in the artfor compositions and methods to specifically boost host anti-viral(e.g., anti-EBV) immune responses, as well as inhibiting hypoxiaassociated angiogenesis in a number of disorders.

SUMMARY OF THE INVENTION

The present invention relates in general to a role of galectin-1 (Gal1)in diagnosing, prognosing, monitoring, treating and/or modulating PTLD,including EBV-associated PTLD and/or hypoxia associated angiogenesisdisorders.

The present inventors have determined a vascular regulatory circuitinvolving Galectin-1 (Gal1), a member of a highly-conserved family ofanimal lectins, is expressed and secreted by a variety of tumors whereit contributes to malignant transformation and metastasis (Paez-Ribes etal. (2009) Cancer Cell 15, 220-231; Liu et al. (2005) Nature Rev Cancer5, 29-41), based on the differential glycosylation of ECs that promotesthe formation of lectin-glycan lattices. These interactions couple tumorhypoxia to VEGFR2-mediated neovascularization through mechanisms thatare independent of HIF-1α and VEGF. The ‘glycosylation signature’ of ECscan be selectively altered by tolerogenic, inflammatory, proliferativeand hypoxic stimuli, which can either enable or hinder formation ofthese lattices. Targeted disruption of Gal1-glycan interactions, throughGal1 blockade or prevention of N-glycan branching, attenuatedhypoxia-driven angiogenesis, while promoting extensive remodeling ofvascular networks and increased influx and expansion of immune effectorcells into the tumor parenchyma. These results underscore novelopportunities for targeting aberrant vascular networks, whilesimultaneously potentiating T cell-mediated antitumor immunity.

The present invention is also based, in part, on the identification ofnovel anti-Gal1 monoclonal antibodies. Accordingly, in one aspect, thepresent invention features a monoclonal antibody, or antigen-bindingfragment thereof, wherein the monoclonal antibody comprises a heavychain sequence with at least about 95% identity to a heavy chainsequence selected from the group consisting of the sequences listed inTable 1 or a light chain sequence with at least about 95% identity to alight chain sequence selected from the group consisting of the sequenceslisted in Table 1. In one embodiment, the monoclonal antibody comprisesa heavy chain CDR sequence with at least about 95% identity to a heavychain CDR sequence selected from the group consisting of the sequenceslisted in Table 1 or a light chain CDR sequence with at least about 95%identity to a light chain CDR sequence selected from the groupconsisting of the sequences listed in Table 1. In another embodiment,the monoclonal antibody, or antigen-binding fragment thereof comprises aheavy chain sequence selected from the group consisting of the sequenceslisted in Table 1 or a light chain sequence selected from the groupconsisting of the sequences listed in Table 1. In still anotherembodiment, the monoclonal antibody, or antigen-binding fragment thereofcomprises a heavy chain CDR sequence selected from the group consistingof the sequences listed in Table 1 or a light chain CDR sequenceselected from the group consisting of the sequences listed in Table 1.In yet another embodiment, the monoclonal antibody, or antigen-bindingfragment thereof is chimeric, humanized, composite, or human. In anotherembodiment, the monoclonal antibody, or antigen-binding fragmentthereof, is detectably labeled, comprises an effector domain, comprisesan Fc domain, is a single-chain antibody, or is a Fab fragment. In stillanother embodiment, the monoclonal antibody, or antigen-binding fragmentthereof, inhibits the binding of commercial antibody to Gal1. In yetanother embodiment, the monoclonal antibody, or antigen-binding fragmentthereof, reduces or inhibits at least one Gal1 activity (e.g., bindingto beta-galacostides) relative to the absence of the monoclonal antibodyor antigen-binding fragment thereof selected from the group consisting.Host cells expressing the described monoclonal antibodies, orantigen-binding fragment thereof, are also contemplated.

In another aspect, the present invention features isolated nucleic acidmolecules that hybridize, under stringent conditions, with thecomplement of a nucleic acid encoding a polypeptide selected from thegroup consisting of the sequences listed in Table 1, or sequences withat least about 95% homology to a nucleic acid encoding a polypeptideselected from the group consisting of the sequences listed in Table 1.In one embodiment, the isolated nucleic acid is comprised within avector. In another embodiment, a host cell comprises

In still another aspect, the present invention features a device or kitcomprising at least one monoclonal antibody or antigen-binding fragmentthereof of the present invention, said device or kit optionallycomprising a label to detect the at least one monoclonal antibody orantigen-binding fragment thereof of the present invention, or a complexcomprising the monoclonal antibody or antigen-binding fragment thereofof the present invention.

In yet another aspect, the present invention features a pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereofof the present invention, in a pharmaceutically acceptable carrier.

In another aspect, the present invention features a method of detectingthe presence or level of a Gal1 polypeptide said method comprisingobtaining a sample and detecting said polypeptide in a sample by use ofat least one monoclonal antibody or antigen-binding fragment thereof ofthe present invention. In one embodiment, the method utilizes at leastone monoclonal antibody or antigen-binding fragment thereof of thepresent invention to form a complex with a Gal1 polypeptide and thecomplex is detected in the form of an enzyme linked immunosorbent assay(ELISA), radioimmune assay (RIA), or immunochemically.

In another aspect, the present invention features a method formonitoring the progression of a disease in a subject, the methodcomprising detecting in a subject sample at a first point in time thelevel of expression of Gal1 using at least one monoclonal antibody orantigen-binding fragment thereof of the present invention; repeating theprevious step at a subsequent point in time; and comparing the level ofexpression of said Gal1 detected in steps a) and b) to monitor theprogression of the disease in the subject. In one embodiment, thesubject has undergone treatment to ameliorate the disease between thefirst point in time and the subsequent point in time.

In still another aspect, the present invention features a method forpredicting the clinical outcome of a subject afflicted with a disease,the method comprising determining the level of expression of Gal1 in apatient sample using at least one monoclonal antibody or antigen-bindingfragment thereof of the present invention; determining the level ofexpression of Gal1 in a sample from a control subject having a goodclinical outcome using at least one monoclonal antibody orantigen-binding fragment thereof of the present invention; and comparingthe level of expression of Gal1 in the patient sample and in the samplefrom the control subject; wherein a significantly higher level ofexpression in the patient sample as compared to the expression level inthe sample from the control subject is an indication that the patienthas a poor clinical outcome.

In yet another aspect, the present invention features a method ofassessing the efficacy of a therapy for a disease in a subject, themethod comprising comparing the level of expression of Gal1 using atleast one monoclonal antibody or antigen-binding fragment thereof of thepresent invention, in a first sample obtained from the subject prior toproviding at least a portion of the therapy to the subject, and thelevel of expression of Gal1 in a second sample obtained from the subjectfollowing provision of the portion of the therapy, wherein asignificantly lower level of expression of Gal1 in the second sample,relative to the first sample, is an indication that the therapy isefficacious for inhibiting the disease in the subject.

In another aspect, the present invention features a method for treatinga subject afflicted with a disease comprising administering at least onemonoclonal antibody or antigen-binding fragment thereof of the presentinvention, such that the subject afflicted with the disease is treated.

In another aspect, the present invention features a method formonitoring the progression of a viral-associated PTLD or hypoxiaassociated angiogenesis disorder in a subject, the method comprisingdetecting in a subject sample at a first point in time the level ofexpression of Gal1; repeating the previous step at a subsequent point intime; and comparing the level of expression of said Gal1 detected atvarious time points to monitor the progression of the viral-associatedPTLD or hypoxia associated angiogenesis disorder in the subject. In oneembodiment, the subject has undergone treatment to ameliorate theviral-associated PTLD or hypoxia associated angiogenesis disorderbetween the first point in time and the subsequent point in time.

In yet another aspect, the present invention features a method ofassessing the efficacy of a test compound for inhibiting aviral-associated PTLD in a subject, the method comprising comparing thelevel of expression of Gal1 in a first sample obtained from the subjectand exposed to the test compound; and the level of expression of Gal1 ina second sample obtained from the subject, wherein the second sample isnot exposed to the test compound, and a significantly lower level ofexpression of Gal1, relative to the second sample, is an indication thatthe test compound is efficacious for inhibiting the viral-associatedPTLD in the subject. In one embodiment, the first and second samples areportions of a single sample obtained from the subject or portions ofpooled samples obtained from the subject.

In another aspect, the present invention features a method forpredicting the clinical outcome of a subject afflicted with aviral-associated PTLD or hypoxia associated angiogenesis disorderpatient, the method comprising determining the level of expression ofGal1 in a patient sample; determining the level of expression of Gal1 ina sample from a control subject having a good clinical outcome; andcomparing the level of expression of Gal1 in the patient sample and inthe sample from the control subject; wherein a significantly higherlevel of expression in the patient sample as compared to the expressionlevel in the sample from the control subject is an indication that thepatient has a poor clinical outcome.

In another aspect, the present invention features a method of assessingthe efficacy of a therapy for a viral-associated PTLD or hypoxiaassociated angiogenesis disorder in a subject, the method comprisingcomparing the level of expression of Gal1 in a first sample obtainedfrom the subject prior to providing at least a portion of the therapy tothe subject, and the level of expression of Gal1 in a second sampleobtained from the subject following provision of the portion of thetherapy, wherein a significantly lower level of expression of Gal1 inthe second sample, relative to the first sample, is an indication thatthe therapy is efficacious for inhibiting the viral-associated PTLD orhypoxia associated angiogenesis disorder in the subject.

In some embodiments of the methods of the present invention, a samplecomprises cells obtained from a subject. In another embodiment, cellsare in a fluid selected from the group consisting of whole blood fluid,serum fluid, plasma fluid, interstitial fluid, cerebrospinal fluid,lymph fluid, saliva, stool, and urine. In still another embodiment, thelevel of Gal1 expression is assessed using a reagent which specificallybinds with a Gal1 protein, polypeptide or protein fragment thereof(e.g., an antibody, an antibody derivative, or an antibody fragment). Inyet another embodiment, the level of Gal1 expression is assessed bydetecting the presence in the sample of a transcribed polynucleotideencoded by a Gal1 polynucleotide (e.g., mRNA or cDNA) or a portion ofsaid transcribed polynucleotide. In another embodiment, the step ofdetecting further comprises amplifying the transcribed polynucleotide.In still another embodiment, the level of Gal1 expression is assessed bydetecting the presence in the sample of a transcribed polynucleotidewhich anneals with a Gal1 polynucleotide or anneals with a portion of aGal1 polynucleotide, under stringent hybridization conditions. In yetanother embodiment, a significant increase comprises an at least twofold or at least five fold increase between the level of expression ofGal1 in the subject sample relative to the normal level of expression ofGal1 in the sample from the control subject.

In another aspect, the present invention features a method formodulating an immune response in a subject afflicted with aviral-associated PTLD or hypoxia associated angiogenesis disordercomprising contacting an immune cell with an agent that modulates theinteraction between Gal1 or a fragment thereof and its natural bindingpartner(s) to thereby modulate the immune response. In one embodiment,the immune response is upregulated. In another embodiment, the immuneresponse is downregulated. In still another embodiment, signaling viathe Gal1 binding partner is inhibited using an agent selected from thegroup consisting of: a blocking antibody or an antigen binding fragmentthereof that recognizes Gal1, a blocking antibody or an antigen bindingfragment thereof that recognizes the Gal1 binding partner(s) or afragment thereof, natural ligands, small molecules, aptamers, peptides,peptidomimetics, glycan-related compounds, glycomimetics, and RNAinterference molecules. In yet another embodiment, the immune cell isfurther contacted (e.g., in vivo or in vitro) with an additional agentthat upregulates an immune response.

In still another aspect, the present invention features a method fortreating a subject afflicted with a viral-associated PTLD or hypoxiaassociated angiogenesis disorder comprising administering an agent thatinhibits the interaction between Gal1 and its natural binding partner(s)on cells of the subject such that the subject afflicted with theviral-associated PTLD is treated. In one embodiment, the agent isselected from the group consisting of: a blocking antibody or an antigenbinding fragment thereof that recognizes Gal1, a blocking antibody or anantigen binding fragment thereof that recognizes the Gal1 bindingpartner(s) or a fragment thereof, natural ligands, small molecules,aptamers, peptides, peptidomimetics, glycan-related compounds,glycomimetics, and RNA interference molecules. In another embodiment, asecond agent that upregulates an immune response, downregulates hypoxiaassociated angiogenesis (e.g., VEGF-targeted therapeutic such as ananti-VEGF antibody), or combination thereof, is administered to thesubject.

In yet another aspect, the present invention features a method formodulating angiogenesis in a hypoxia associated angiogenesis disordercomprising contacting a cell exhibiting hypoxia associated angiogenesiswith an agent that modulates the interaction between Gal1 or a fragmentthereof and its natural binding partner(s) to thereby modulateangiogenesis. In one embodiment, hypoxia associated angiogenesis isdownregulated. In another embodiment, downregulation of hypoxiaassociated angiogenesis is determined by at least one effect selectedfrom the group consisting of: reduction in vessel diameter, reduction invessel distribution, reduction in tortuous vessels, increase in pericytecoverage, increase in the fraction of pericytes that are mature, andreduction in pimonidazole adduct formation. In still another embodiment,hypoxia associated angiogenesis is modulated using an agent selectedfrom the group consisting of: a blocking antibody or an antigen bindingfragment thereof that recognizes Gal1, a blocking antibody or an antigenbinding fragment thereof that recognizes a Gal1 binding partner(s) or afragment thereof, natural ligands, small molecules, aptamers, peptides,peptidomimetics, glycan-related compounds, glycomimctics, and RNAinterference molecules. In yet another embodiment, the method furthercomprises contacting the cell (e.g., in vivo or in vitro) exhibitinghypoxia associated angiogenesis with an additional agent thatdownregulates hypoxia associate angiogenesis.

In another aspect, the present invention features an isolated complexcomprising a Gal1 polypeptide and a VEGFR2 polypeptide. In oneembodiment, the Gal1 polypeptide is a polypeptide describe herein orfragment thereof that is capable of binding to a VEGFR2 polypeptide andthe VEGR2 polypeptide described herein or fragment thereof that iscapable of binding to a Gal1 polypeptide. In another embodiment, atleast one polypeptide or fragment is a fusion protein. In still anotherembodiment, at least one polypeptide or fragment is labeled. In yetanother embodiment, the complex is generated within a host cell. Inanother embodiment, the Gal1 polypeptide or fragment thereof and saidVEGFR2 polypeptide or fragment thereof are covalently linked.

In still another aspect, the present invention features an isolatedantibody of the present invention has the ability to disrupt a complexcomprising a Gal1 polypeptide and a VEGFR2 polypeptide.

In yet another aspect, the present invention features a method foridentifying a compound that modulates a Gal1/VEGFR2 complex comprising:a) contacting the complex with a test compound; and b) assaying theamount or activity of the complex, wherein a change in the amount oractivity of the complex in the presence of the test compound as comparedto the amount or activity of the complex in the absence of the testcompound is indicative of a compound that modulates a Gal1/VEGFR2complex.

In another aspect, the present invention features a hybridoma, 14-198F4-F8-G7, deposited under accession number PTA-10535.

For any method described herein, the relevant condition, disease, ordisorder can be a viral-associated PTLD, cancer, and/or a hypoxiaassociated angiogenesis disorder. In addition, the level of expressionof a marker, such as Gal1 to be analyzed, can be determined by theamount, structure, subcellular localization, and/or activity of themarker, as described further herein. Moreover, the progress, outcome, orefficacy of any method describe herein can be measured by at least onecriteria selected from the group consisting of survival until mortality,pathological complete response, clinical complete remission, clinicalpartial remission, clinical stable disease, recurrence-free survival,metastasis free survival, and disease free survival, according to, butnot limited by, exemplary embodiments described further herein. Also,the Gal1 binding partner can be VEGFR2 for any method, composition,and/or complex of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows serial dilution-based reactivity data for anti-human Gal1monoclonal antibodies assayed against endogenous Gal1 from a Hodgkinlymphoma cell line.

FIG. 2 shows cross-reactivity data for anti-human Gal1 monoclonalantibodies assayed against endogenous cynomologous monkey and mouseGal1.

FIG. 3 shows a schematic diagram of GST-tagged human Gal1 (hGal1)fragments utilized in epitope mapping analyses of the anti-human Gal1monoclonal antibodies.

FIG. 4 shows epitope mapping data for anti-human Gal1 monoclonalantibodies.

FIG. 5 shows Gal1 transcript abundance in EBV-transformed lymphoblastoidcell lines.Gal1 transcript abundance in HL lines, LCLs, normal B-cellsand additional B-cell neoplasmswas assessed using publically availablegene expression profiles (Kuppers R. (2009) Nat Rev Cancer 9:15-27).Color scale at the bottom indicates relative expression±SEM. Redconnotes high-level expression; blue indicates low-level expression.

FIG. 6 shows specificity of the anti-Gal1 monoclonal antibody, 8F4F8G7,for endogenous Gal1. The newly developed Gal1 mAb specifically detectedendogenous Gal1 from the cHL cell line, L428, on immunoblots.

FIGS. 7A-7B shows Gal 1 expression in EBV-transformed LCLs and EBV+primary PTLDs. FIG. 7A shows Gal1 expression in a cHL cell line (L428),a series of EBV-transformed LCLs (NOR-, RIC-, STA-, FOL-, LOV-, RIV-,WOL-, FW-, VS-, MA-, SC-, DS-, AND DW-LCL), and a DLBCL cell line(SU-DHL6). FIG. 7B shows Gal1 immunohistochemical staining of threerepresentative primary EBV+PTLDs (panels a, b, and c) and a DLBCL (paneld). The recently developed murine αGal1mAb, 8F4F8G7, was used at1:20,000 in immunoblots in FIG. 7A and 1:40,000 in IHC in FIG. 7B.Original magnifications: ×1000.

FIG. 8 shows Gal1 expression in primary post-transplantlymphoproliferative disorders (PTLDs) and DLBCLs. Gal1immunohistochemistry (IHC) was performed with the previously describedrabbit anti-Gal1 polyclonal antiserum (Juszczynski et al. (2007) ProcNatl Acad Sci USA 104:13134-9). Representative primary EBV+PTLDs (panelsa, b, and c) and DLBCLs (panels d, c, and f) are shown. Originalmagnifications: ×1000.

FIGS. 9A-9E show AP-1 dependent Gal1 expression in EBV-transformed LCLsand primary PTLDs. FIG. 9A shows total phospho-cJun and JunB expressionin a cHL cell line, L428, and two EBV-transformed LCLs, RIC andNOR-actin was used as a loading control. FIG. 9B shows results ofChIP-PCR analysis of cJun and JunB binding to Gal1 enhancer regions inthe cHL cell line, L428, and two LCLs, NOR and RIC. Results arerepresentative of triplicate experiments. FIG. 9C shows results ofdensitometric analyses of ChIP-PCR data from FIG. 9B. FIG. 9D shows Gal1promoter and enhancer-driven luciferase activity in LCLs. NOR cells werecotransfected with 300 ng of the pGL3-Gal 1-promoter constructs (withoutor with the wild-type or mutant AP-1 dependent Gal1 enhancer) and 100 ngof the control reporter plasmid, pRL-TK, and evaluated for relativeluciferase activity as described (Juszczynski et al. (2007) Proc NatlAcad Sci USA 104:13134-9). FIG. 9E shows IHC analysis of JunB (panels a,c, and e) and phospho-cJun (panels b, d, and f) in 3 primary PTLDs. ThePTLDs had uniformly high nuclear staining of JunB and positivephospho-cJun staining of variable intensity.

FIGS. 10A-10B show Gal1 transcript abundance in LMP1-expressing CD10+germinal center B cells. Gal1 transcript abundance in normal CD10⁺germinal center B-cells with and without LMP1 transduction was assessedusing publically available gene expression profiles (Vockerodt et al.(2008) J Pathol (2008) 216:83-92). Gal1 induction is shown on a heatmap, with red indicating high expression and blue indicating lowexpression in FIG. 10A. FIG. 10B shows that Gal1 was 2 fold moreabundant in LMP-transduced germinal center B cells (p<0.002).

FIGS. 11A-11C show induction of Gal1 expression by LMP1 and 2A. FIG. 11Ashows LMP1- and LMP2A-enhanced Gal1 promoter-driven luciferase activity.293T cells were co-transfected with the pGL3-LGALS1 promoter(Juszczynski et al. (2007) Proc Natl Acad Sci USA 104:13134-9), controlreporter plasmid pRL-PGK and pFLAG-CMV2 empty vector or expressionvector LMP1-FLAG or LMP2A-FLAG or LMP1-FLAG plus LMP2A-FLAG andevaluated for relative luciferase activity. FIG. 11B shows RNAi-mediateddown-regulation of LMP2A in EBV-transformed LCL. NOR. β-actin was usedas a loading control. FIG. 11C shows chemical inhibition of PI3Kactivity (25 μM Ly294002) and associated change in Gal1 expression inEBV-transformed LCLs.

FIG. 12 shows regulatory element analysis of the Gal1 promoter. Analysisof transcription factor binding motifs and modules within the Gal1promoter region revealed a single NFκB binding site, and a NFAT/NF-Ymodule. The Gal1 promoter region included in the luciferase constructs(FIGS. 11A-11C) is shown in blue relative to the transcription startsite (TSS).

FIGS. 13A-13B show the anti-Gal1 mAb 8F4F8G7 inhibits rGal1-inducedapoptosis of in vitro activated T cells. Anti-CD3/CD28 activated human Tcells were treated with 10 μM of rGal1 alone or 10 μM rGal1pre-incubated with 5 μM of anti-Gal1 mAb (8F4F8G7) or an isotype-matchedIgG2b control and evaluated thereafter with a flow cytometric apoptosisassay (Annexin V-FITC and PI staining). The percentage of cells in eachquadrant is indicated in FIG. 13A. FIG. 13B shows a histogramsummarizing the percentage of annexin V+ cells in the absence of rGal orthe presence of rGal alone or rGal1 pre-incubated with the αGal1 mAb orisotype control.

FIGS. 14A-14B show that Gal1 neutralizing mAb, 8F4F8G7, inhibitsGal1-mediated apoptosis of EBV-specific CTLs. FIG. 14A shows results ofEBV-specific CTLs treated with rGal1 alone or rGal1 pre-incubated withαGal1 mAb or isotype control. The percentage of viable CD8+ CTLs (7-AADnegative) is shown on the top of the gate. FIG. 14B shows a histogramsummarizing the percentage of viable EBV-specific CD8+ CTLs followingthe indicated treatments.

FIGS. 15A-15B show results of αGal mAb-mediated inhibition ofrGal1-mediated apoptosis of EBV-specific CTLs generated from a secondindependent donor. FIG. 15A shows results of EBV-specific CTLs treatedwith rGal1 alone or rGal1 pre-incubated with anti-Gal1 mAb or isotypecontrol IgG2b as in FIGS. 14A-14B. The percentage of viable CD8+ CTLs(7-AAD negative) is shown on the top of the gate. FIG. 15B shows ahistogram summarizing the percentage of viable CD8+ CTLs following theindicated treatments.

FIGS. 16A-16S show that differential glycosylation of endothelial cells(ECs) controls the formation of lectin-glycan lattices. FIG. 16A showsthe glycan repertoire of HUVEC under resting conditions (2% FCS)detected with biotinylated L-PHA, LEL, SNA, MAL II, PNA and HPA (filledhistograms) or with PE-conjugated stravidin alone (open histograms).Data are representative of eight independent experiments. FIG. 16B showsthe glycan repertoire of HUVEC under resting, proliferative (bFGF),tolerogenic (IL-10 and/or TGF-β₁) or pro-inflammatory (TNF, IFN-γ and/orIL-17) conditions. rMFI (relative mean fluorescence intensity)=(MFI withlectin−MFI without lectin)/MFI without lectin. Data are presented as theratio relative to resting conditions (dotted line; value=1) and are themean±SEM of four independent experiments. FIG. 16C shows binding resultsof 488-Gal1 to HUVEC with or without lactose or sucrose, swainsonine orbenzyl-α-GalNAc. Data are the mean±SEM of three independent experiments.FIG. 16D shows binding results of 488-Gal1 to HUVEC transfected withGnT5 or GCNT1 siRNA. Cells without siRNA or transfected with scrambled(src) siRNA were used as controls. Data are the mean±SEM of at leastthree independent experiments. FIG. 16E shows binding results of488-Gal1 to HUVEC exposed to tolerogenic, proliferative or inflammatorystimuli. Data are presented as the rMFI ratio relative to resting ECs(dotted line; value=1) and are the mean±SEM of four independentexperiments. * P<0.05, ** P<0.01 versus control. FIGS. 16F-16H showresults of [³H]thymidine incorporation (FIG. 16F), migration (FIG. 16G)and tube formation (FIG. 16H) of ECs transfected or not with GnT5, GCNT1or scr siRNA and treated or not with Gal1 (1 μM) and/or VEGF (20 ng/ml)with or without lactose. P<0.05 vs Gal1; *P<0.05 ** P<0.01 versuscontrol. Data are the mean±SEM of at least five independent experiments.FIG. 16I shows tube formation induced by Gal1 or VEGF in HUVECtransfected with GnT5, GCNT1 or scr siRNA. * P<0.05 versus scr siRNA.Data are the mean±SEM of three independent experiments. FIG. 16J showsin vivo vascularization of Matrigel sponges containing Gal1 with orwithout lactose and the right panel in particular shows quantificationof hemoglobin content. Data are representative of two independentexperiments. FIG. 16K shows schematic representation of N- and O-glycanbiosynthesis, including relevant glycosyltransferases, such as α2-6sialyltransferase 1 (ST6Gal1), N-acetylglucosaminyltransferase 5 (GnT5),α2-3 sialyltransferase 1 (ST3Gal1) and core 2N-acetylglucosaminyltransferase 1 (GCNT1), the coordinated actions ofwhich lead to the generation or masking of common glycosylated ligandsfor galectins (N-acetyllactosamine; LacNAc) or poly-LacNAc residues incomplex N-glycans or core 2 O-glycans) at the top, whereas the bottomshows schematic representation of lectin-binding sites in N- andO-glycans. Specific residues recognized by MAL II, LEL, SNA and L-PHA oncomplex N-glycans and by HPA, PNA and LEL on O-glycans are indicated(green). The common glycosylated ligand for Gal1 (LacNAc) is alsoindicated (purple). FIG. 16L shows binding results of biotinylated L-PHAto HUVEC transfected with GnT5 (filled histogram) or with scrambled(scr) (open black histogram) siRNA. Cells stained with PE-conjugatedStravidin alone were used as negative control (open grey histogram).Data are representative of four independent experiments. FIG. 16M showsresults of qRT-PCR analysis of GnT5 mRNA, whereas FIG. 16N shows thatfor GCNT1 mRNA of HUVEC transfected with different concentrations ofspecific siRNA relative to RN18S1 mRNA (AU: arbitrary units). **P<0.01versus control. Data are the mean±SEM of four independent experiments.FIGS. 16O-16Q show dose-dependent proliferation (FIG. 16O), migration(FIG. 16P) and tube formation (FIG. 16Q) of HUVEC incubated with orwithout different concentrations of Gal1, VEGF (20 ng/ml) or both. Gal1effects were completely prevented by co-incubation with 30 mM lactose. *P<0.05 and ** P<0.01, versus control; ^(†) P<0.05 vs Gal1 (1 μM). Dataare the mean±SEM of five experiments. FIG. 16R shows light microscopyimages of capillary tube formation (upper panels) and migration (lowerpanels) of HUVEC incubated with Gal1 in the presence or absence oflactose. VEGF was used as positive control. Images representative offive independent experiments are shown. FIG. 16S shows dose-dependentinvasion of HUVEC in the presence or absence of different concentrationsof Gal1 or VEGF (20 ng/ml). Results are plotted as invasion indexcalculated as the number of fluorescent invasive cells relative tocontrol. * P<0.05 and ** P<0.01. Data are the mean±SEM of fiveexperiments.

FIGS. 17A-17R show the galectin-1 co-opts VEGFR2 signaling pathwaysthrough the formation of lectin-glycan lattices on highly branchedcomplex N-glycans. FIG. 17A shows results of a phospho-RTK signalingarray of HUVEC exposed to medium (control), VEGF or Gal1, wherein in theleft panel, arrows indicate proteins with increased phosphorylationintensity. Data are representative of three independent experiments. Bycontrast, the right panel shows quantification of pixel intensity. *P<0.05, ** P<0.01 versus control. Data are the mean±SEM of threeindependent experiments. FIG. 17B shows immunoblot results of VEGFR2,Akt and Erk1/2 phosphorylation in HUVEC treated with differentconcentrations of Gal1. Data are representative of six independentexperiments. FIGS. 17C-17E shows Gal1-induced proliferation (FIG. 17C),migration (FIG. 17D) and tube formation (FIG. 17E) on HUVECpre-incubated with pharmacological inhibitors of PI(3)K/Akt (LY294002),Erk1/2 (U0126), JAK2-STAT3 (AG490), Jnk/SAP (SP600125), p38 (SB202190)or NF-κB (BAY11-7082). ** P<0.01 versus Gal1. Data are the mean±SEM offive independent experiments. FIG. 17F shows immunoblot analysis ofVEGFR2, Akt and Erk1/2 phosphorylation induced by Gal1 or VEGF in HUVECtransfected with VEGFR2 or GnT5 siRNA. Data are representative of threeindependent experiments. FIG. 17G shows co-immunoprecipitation resultsfollowed by immunoblot analysis of HUVEC lysates, wherein the left panelshows results from cells treated with or without Gal1 and the rightpanel shows results from cells transfected or not with GnT5 or GCNT1siRNA or exposed to PNGase F and treated with Gal1. Input, whole celllysate; IB, immunoblot; IP, immunoprecipitation. Data are representativeof three independent experiments. FIG. 17H shows laser confocalmicroscopy results of HUVEC transfected or not with GnT5 siRNA andtreated with Gal1 or buffer control stained for VEGFR2 (red) or fornuclei (DAPI; blue). Images are representative of four independentexperiments are shown. FIG. 17I shows tube formation results of HUVECtransfected or not with VEGFR2, NRP-1, VEGF or scr siRNA treated or notwith Gal1. * P<0.05 versus Gal1. Data are representative of threeindependent experiments. FIG. 17J shows tube formation results of HUVECpre-treated with lactose or blocking antibodies to VEGFR1, VEGFR2,VEGFR3 or VEGF. * P<0.05 versus Gal1. Data are representative of threeindependent experiments. FIG. 17K shows fold increase results in thephosphorylation status of a panel of growth factor receptor tyrosinekinases (RTKs) and signaling nodes as determined by phospho-RTKsignaling array upon exposure of HUVEC to Gal1 or VEGF. The relativesignal intensity of each spot, quantified as pixel intensity isrepresented relative to control intensity (value=1, dotted line). *P<0.05; ** P<0.01 versus control. Data are the mean±SEM of threeindependent experiments. FIG. 17L shows immunoblot analysis results ofVEGFR2 and FIG. 17M shows immunoblot analysis results of NRP-1 in HUVECtransfected with specific siRNA (100 nM). Data are representative ofthree independent experiments. FIG. 17N shows co-immunoprecipitationresults followed by immunoblot analysis of cell lysates derived fromHUVEC cultured with or without Gal1. Input, whole cell lysate; IB,immunoblot; IP, immunoprecipitation. Data are representative of threeindependent experiments. FIG. 2O shows ELISA results of VEGF secretionby HUVEC after specific siRNA-mediated silencing. nd, not-detected. Dataare the mean±SEM of four experiments. FIGS. 17P-17Q show migrationresults of HUVEC induced by Gal1 or VEGF in transwells. Cells weretransfected with 100 nM siRNA specific for VEGFR2, NRP-1 or VEGF (FIG.17P), or were incubated with specific blocking antibodies to VEGFR2 orVEGF (FIG. 17Q). * P<0.05, ** P<0.01 versus medium, Gal1 or VEGF alone.Data are representative of three independent experiments. FIG. 17R showsELISA results of VEGF secretion by HUVEC incubated with differentconcentrations of Gal1 with or without lactose. Hypoxia was used aspositive control of VEGF secretion. Data are the mean±SEM of sixindependent experiments.

FIGS. 18A-18U show the galectin-1-glycan lattices link tumor hypoxia toVEGFR2-mediated angiogenesis. FIG. 18A shows the glycan repertoire onHUVEC incubated in hypoxia (black filled histograms) or normoxia (greyfilled histograms), detected with biotinylated L-PHA, LEL, SNA, MAL IIor PNA, or with PE-conjugated stravidin alone (open histograms). Dataare representative of five independent experiments. FIG. 18B showsbinding results of 488-Gal1 to HUVEC exposed to hypoxia or normoxia. **P<0.01. Data are the mean±SEM of five independent experiments. FIGS.18C-18F show expression of Gal1 in KS cells transfected with or withoutHIF-1α siRNA or a super-repressor form of IκB-α (IκB-α-SR) and incubatedunder hypoxia or normoxia. FIG. 18C shows promoter activity and data arethe mean±SEM of five independent experiments. FIG. 18D shows qRT-PCRresults of Gal1 mRNA relative to RN18S1. AU, arbitrary units. **P<0.01.Data are the mean±SEM of three independent experiments. FIG. 18E showsimmunoblot results of Gal1, IκB-α and HIF-1α. Data are representative offour experiments. FIG. 18F shows ELISA results of Gal1 secretion.**P<0.01. Data are the mean±SEM of three independent experiments. FIG.18G shows ELISA results of Gal1 secretion by KS cells cultured inhypoxia or normoxia in the presence or absence of N-acetyl-cysteine(NAC; 0.5 mM). FIG. 18H shows ELISA results of Gal1 secretion by KScells exposed to H₂O₂ (0.5 mM) in the presence or absence of BAY11-7082. Data are the mean±SEM of three independent experiments. FIG.18I shows immunoperoxidase staining results of Gal1 in non-hypoxic andhypoxic areas of KS xenografts in the upper panels, whereas the lowerpanels show immunofluorescence of Gal1 and Hypoxyprobe-1 staining.Images are representative of three independent experiments. FIG. 18Jshow tube formation results by HUVEC incubated with conditioned medium(CM) from normoxic or hypoxic KS cells transfected or not with scr orVEGF siRNA and/or Gal1 shRNA. ** P<0.01. Data are the mean±SEM of fourindependent experiments. FIG. 18K shows hemoglobin content results ofMatrigel plugs containing CM of KS cells transfected or not with Gal1 orscr shRNA, cultured under hypoxic or normoxic conditions and inoculatedinto wild-type or Lgals1^(−/−) mice. ** P<0.01. Data are the mean±SEM offour independent experiments. FIG. 18L shows tube formation results byHUVEC transfected with GnT5, GCNT1 or scr siRNA incubated with CM fromnormoxic or hypoxic KS cells. ** P<0.01. Data are the mean±SEM of fourindependent experiments. FIG. 18M shows immunoblot analysis of Gal1expression induced by hypoxia in human and mouse melanoma (A375 andB16-F0), mouse breast carcinoma (4T1) and human prostate carcinoma(LNCaP) cell lines. Right panel, quantification of band intensityrelative to that of actin. Data are representative of three independentexperiments. FIG. 18N shows secretion of Gal1 by KS cells cultured underhypoxic or normoxic conditions in the presence or absence of HIF-1α orNF-κB inhibitor. ** P<0.01. Data are the mean±SEM of three independentexperiments. FIG. 18O shows expression results of Gal1 upon treatment ofKS cells with CoCl₂ (chemical activator of HIF-1α) evaluated byimmunoblot (left panel) or promoter activity (right panel) assays.Modulation of pGL3-Gal1-Luciferase activity relative to renillaexpression is shown. ** P<0.01. Data are the mean±SEM of threeindependent experiments. FIG. 18P shows putative NF-κB consensussequences (SEQ ID NOS 28-35, respectively, in order of appearance)revealed by in silico analysis (MatInspector Software) of the regulatorysequences of human LGALS1 gene. A fragment ranging from 2400 bp upstreamto 2500 bp downstream from the start site (+1) of LGALS1 coding sequencewas analyzed. A relevant NF-κB consensus sequence (#3) located at thepromoter sequence 341 by upstream of the start site is highlighted. Aschematic representation of the LGALS1 gene fragment indicating theeight putative NF-κB consensus sequences is shown. A schematicrepresentation of pGL3-Gal1-Luc, used in luciferase assays, whichconsists of LGALS1 promoter region (−473 to +67, encompassing NF-κBconsensus sequence #3) ligated into the pGL3 promoterless reportervector is shown. FIG. 18Q shows ELISA results of Gal1 secretion andimmunoblot analysis of Gal1 and IκB-α expression (inset) by KS cellscultured in hypoxia or normoxia in the presence or absence of increasingconcentrations of the ROS scavenger NAC. * P<0.05; ** P<0.01 versuscontrol. Data are the mean±SEM. of three independent experiments. FIG.18R shows ELISA results of Gal1 secretion by KS cells cultured withincreasing concentrations of H₂O₂. Data are the mean±SEM of threeindependent experiments. FIG. 18S shows immunoblot results of KS cellsexpressing shRNA constructs that target different sequences of humanGal1 mRNA (sh-Gal1.1, sh-Gal1.2 and sh-Gal1-3) or scrambled shRNA(sh-scr) cultured under normoxic (upper panel) or hypoxic (lower panel)conditions. rGal1, recombinant Gal1. The lower right panel shows laserconfocal microscopy results of sh-Gal-1.2 KS cells co-infected withGFP-encoding vector fixed and stained with anti-Gal1 antibody (red).Data are representative of five independent experiments. FIGS. 18T-18Ushow ELISA results of VEGF (FIG. 18T) or Gal1 (FIG. 18U) secretion bysh-scr or sh-Gal1.2 KS cells transfected with 100 nM siRNA specific forVEGF (VEGF siRNA) or scr siRNA incubated under normoxic or hypoxicconditions. Data are the mean±SEM of four independent experiments.

FIGS. 19A-19N show that targeting galectin-1-glycan lattices in vivoprevents tumor growth and angiogenesis. FIGS. 19A-19C show results ofnude mice inoculated with KS clones (5×10⁶ cells) expressing Gal1 shRNA(sh-Gal1.1 and sh-Gal1.2), control KS cells expressing scr shRNA(sh-scr) or wild-type KS cells (KS wt). * P<0.05, ** P<0.01 versussh-scr. Data are the mean±SEM of four independent experiments with fiveanimals per group. FIG. 19A shows results of tumor growth. FIG. 19B showresults of flow cytometry of tumor-associated CD34⁺ ECs. Dot plots arerepresentative of four independent experiments. FIG. 19C shows resultsof tumor hemoglobin content. FIG. 19D shows Gal1 transcript profiles ofmouse mECK36 KS tumors compared to normal skin in the left panel,whereas the right panel shows laser confocal microscopy of mECK36stained for Gal1 and LANA. FIG. 19E shows a Gal1 transcript profile ofhuman KS compared to normal skin. FIG. 19F shows representative imagesof human benign vascular lesions (n=26) and primary KS tumors (n=15)stained with H&E or with anti-Gal1 antibody, wherein quantification ofGal1 expression is shown to the right. ** P<0.01. FIG. 19G shows resultsof in vitro cell growth of KS clones expressing Gal1 shRNA (sh-Gal1.1and sh.Gal1.2), scr shRNA (sh-scr) or wild-type KS cells (KS wt). Dataare the mean±SEM of four independent experiments. FIG. 19H shows flowcytometry results of tumor-associated CD34⁺ ECs of nude mice inoculatedwith KS clones. Data are the mean±SEM of three independent experiments.** P<0.01 versus sh-scr. FIG. 19I shows immunoblot results of KS clonesgenerated by limited dilution of antisense transfectants (As-Gal1.1,As-Gal1.2 and As-Gal1.3) or wild type KS cells (KS wt). Data arerepresentative of three experiments. FIG. 19J shows in vitro cell growthresults of KS wt cells, control KS cells transfected with vector alone(As-control) and Gal1 knockdown KS clones. Data are the mean±SEM ofthree independent experiments. FIGS. 19K-19M show results of nude miceinoculated with As-Gal1.1, As-Gal1.2, As-Gal1.3, As-control or wt KScells. FIG. 19K shows kinetics of tumor growth. P<0.05. Data are themean±SEM of three independent experiments with three animals per group.FIG. 19L shows quantitative analysis of tumor microvessel density. *P<0.05. Data are the mean±SEM of three independent experiments withthree animals per group. FIG. 19M shows qRT-PCR results of Gal1 mRNA inmECK36 KS tumors and normal skin. ** P<0.01. FIG. 19N showsrepresentative images of human benign vascular lesions (n=26) andprimary KS tumors (n=15) stained with H&E or with anti-Gal1 antibody.

FIGS. 20A-20J show that targeted disruption of galectin-1-glycanlattices in vivo targets both vascular and immune compartments. FIGS.20A-20F show results from B6 mice inoculated with B16 clones (2×10⁵cells) expressing Gal1 shRNA (sh-Gal1.1 and sh-Gal1.2), sh-scr orwild-type B16 cells (B16 wt). For FIGS. 20A-20C, * P<0.05, ** P<0.01versus sh-scr, whereas for FIGS. 20D-20F, ** P<0.01 versus sh-scr. Dataare the mean±SEM of three independent experiments. FIG. 5A shows thekinetics of tumor growth. FIG. 20B shows the results of flow cytometryof tumor-associated CD34⁺ ECs. FIG. 20C shows tumor hemoglobin content.FIG. 20D shows proliferation and FIG. 20E shows secretion of IFN-γ andIL-17 by TDLN cells from mice receiving B16 knockdown clones or controltransfectants after ex vivo restimulation with B16 cells. nd, notdetected. FIG. 20F shows flow cytometry results ofCD4⁺CD25⁺FoxP3⁺T_(reg) cells in TDLN from mice receiving knockdownclones or control transfectants. FIG. 20G shows confocal microscopyresults of lectin staining (green) and CD31⁺ ECs (red) in B16 tumors andnormal skin, wherein the left panel shows quantification of fluorescenceintensity (10 fields per tumor, 200×). Mean represents the ratio ofgreen versus red fluorescence. FIG. 20H shows IHC of biopsies (n=19)from patients with primary melanoma stained with anti-Gal1 or anti-CD31antibodies, wherein representative images are shown and the right panelshows the correlation between Gal1 expression and microvascular density(MVD). FIG. 20I shows immunoblot results of B16 clones expressing Gal1shRNA (sh-Gal1.1 and sh-Gal1.2), control B16 cells expressing scr shRNA(sh-scr) or wild-type B16 cells (B16 wt). Data are representative ofthree experiments. FIG. 20J shows in vitro cell growth of B16 clonesexpressing Gal1 shRNA (sh-Gal1.1 and sh.Gal1.2), scr shRNA (sh-scr) orwild-type B16 cells (B16 wt). Data are the mean±SEM of three independentexperiments.

FIGS. 21A-21K show that mAb-mediated galectin-1 blockade modulatesvascular biology and attenuates abnormal angiogenesis in vivo. FIG. 21Ashows results of binding of 488-Gal1 to HUVEC in the presence or absenceof 8F4F8G7 mAb (0.5 μM), isotype control (Iso) or lactose. The filledhistogram shows non-specific binding determined with unlabeled Gal1.Data are representative of four independent experiments. FIGS. 21B-21Dshow the functional activity of 8F4F8G7 mAb in vitro (** P<0.01 versusisotype. Data are the mean±SEM for FIGS. 6B-6D or are representative ofthree independent experiments for FIG. 6E.) FIG. 21B showsproliferation, FIG. 21C shows migration, and FIG. 21D shows tubeformation of HUVEC incubated with Gal1 or VEGF in the presence orabsence of 8F4F8G7 mAb or isotype control. FIG. 21E shows immunoblotresults of VEGFR2 phosphorylation induced by Gal1 in HUVEC incubatedwith 8F4F8G7 mAb or isotype control or in HUVEC transfected with GnT5siRNA. FIGS. 21F-21H show the results of nude mice inoculated withwild-type KS treated in vivo with 8F4F8G7 mAb (7.5 mg/kg) or isotypecontrol every three days (* P<0.05 versus isotype. Data are the mean±SEMof four independent experiments with five animals per group). FIG. 21Fshows the kinetics of tumor growth. FIG. 21G shows the results of flowcytometry of tumor-associated CD34⁺ ECs. FIG. 21H shows tumor hemoglobincontent. FIG. 21I shows binding results of fluorescently-labeled(488)-Gal1 to HUVEC in the presence or absence of 8F4F8G7, 8B5E6H9 or2E52H12 anti-Gal1 mAb (all used at 0.5 μM). * P<0.05 versus control.Data are the mean±SEM of three independent experiments. FIG. 21J showsbinding results of 488-Gal3 (20 μg/ml, left panel) and 488-Gal8 (20μg/ml, right panel) to HUVEC in the presence or absence of 8F4F8G7 mAb(0.5 μM). Filled histogram show non-specific binding determined withunlabeled galectins. Data are representative of three independentexperiments. FIG. 21K shows tumor growth results in nude mice inoculatedwith wild-type KS cells and treated in vivo every three days withdifferent doses of 8F4F8G7 mAb or with isotype control. Data are themean±SEM of three independent experiments. * P<0.05 versus isotypecontrol.

FIGS. 22A-22R show that therapeutic administration of a neutralizinganti-galectin-1 mAb promotes vascular remodeling and tumor-specificimmunity. FIGS. 22A-22J show the results of B6 mice inoculated with2×10⁵ wild type B16 cells treated in vivo with 8F4F8G7 mAb (7.5 mg/kg)or with isotype control every three days. FIG. 22A shows kinetics oftumor growth. * P<0.05, ** P<0.01 versus B16. Data are the mean±SEM offour independent experiments with six animals per group. FIG. 22B showsconfocal microscopy results of lectin (GLS-1_(B4))-perfused vessels insized-matched tumors. FIG. 22C shows quantification of vessel diameters(10 fields per tumor, 200×). FIG. 22D shows confocal microscopy resultsof lectin-perfused vessels (green) labeled with anti-αSMA antibody(red). Arrows indicate vessel-associated pericytes, wherein the rightpanel shows the percentage of tumor vessels with pericyte coverage (10fields per tumor, 200×). FIG. 22E shows confocal microscopy results oftumors stained with anti-desmin (red, upper panels) or anti-RGS5 (red,lower panels). ECs were stained with anti-CD31 (green) andquantification of vessels covered by pericytes expressing RGS5, desmin,αSMA and PDGFRβ is shown to the right. For FIGS. 22C-22E, **P<0.01versus isotype control and data are the mean±SEM of three independentexperiments with four animals per group. FIG. 22F shows confocalmicroscopy results of B16 sized-matched tumors immunostained withHypoxyprobe-1. FIG. 22G shows proliferation results and FIG. 22H showssecretion results of IFN-γ and IL-17 (FIG. 22H) by TDLN cells from micetreated with 8F4F8G7 mAb or isotype control in response to ex vivorestimulation with B16 cells, wherein **P<0.01 versus isotype controland data are the mean±SEM of four independent experiments with four miceper group for both figures. FIG. 22I shows flow cytometry results ofCD25⁺FoxP3⁺TDLN cells from mice given 8F4F8G7 mAb or isotype control.Dot plots are representative of four independent experiments. FIG. 7Jshows confocal microscopy results of tumor infiltrating-CD8⁺ T cells inthe left panel, whereas the right panel shows flow cytometry results ofIFN-γ-expressing tumor infiltrating-CD8⁺ T cells. Data are the mean±SEMof three independent experiments with four mice per group. FIG. 22Kshows results of spleen T cells purified from B16 tumor-bearing mice,stained with CFSE and transferred (5×10⁶) to mice with establishedsyngeneic tumors treated with 8F4F8G7 mAb or with isotype control.Representative dot plots of CFSE T cells reaching tumors and spleen ofrecipient mice are shown. The number at the top right of the figureindicates positive events. FIG. 22L shows the number offluorescently-labeled beads (relative to 1×10⁵ events) reaching tumorsand spleen of mice given 8F4F8G7 mAb or isotype control 15 min afterinoculation. Data are the mean±SEM of two independent experiments withfour animals per group. **P<0.01 versus isotype control. FIG. 22M showstumor growth results in B6-Rag1^(−/−) immunodeficient mice inoculatedwith 2×10⁵ wild type B16 cells treated in vivo with 8F4F8G7 mAb (7.5mg/kg) or with isotype control every three days. * P<0.05 versus isotypecontrol. Data are the mean±SEM of two independent experiments with fouranimals per group. FIG. 22N-22Q show results of immunocompetent B6 miceinoculated with 2×10⁵ wild-typeB16 cells treated in vivo with 8F4F8G7mAb (7.5 mg/kg) or with isotype control every three days. FIG. 22N showsflow cytometry results of tumor-associated CD34⁺ ECs. P=N.S. at day 20after tumor inoculation. FIG. 22O shows laser confocal microscopyresults of tumors immunostained with anti-Rgs5 (red) or anti-desmin(red). ECs were stained with anti-CD31 (green). FIG. 22P shows flowcytometry results of IFN-γ-, IL-17- and IL-10-producing CD4⁺ T cells inTDLN from mice treated with 8F4F8G7 mAb or isotype control in responseto ex vivo restimulation with B16 cells. Numbers in the top rightquadrants indicate percentage of double positive cells. Data arerepresentative of three independent experiments with four mice pergroup. FIG. 22Q shows flow cytometry results of FoxP3 expression withinCD4⁺CD25⁺cells in TDLN of B16 tumors from mice treated with 8F4F8G7 mAbor isotype control. Data are the mean±SEM of three independentexperiments. FIG. 22R shows results of spleen T cells isolated from B16tumor-bearing mice and stained with CFSE inoculated (5×10⁶) in mice withestablished syngeneic tumors treated with the 8F4F8G7 mAb or withisotype control. The number of CFSE⁺ cells/0.1 cm³ in tumors and spleenof recipient mice is shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery thatgalectin-1 (Gal1) is overexpressed by viral-associatedpost-transplantation lymphoblastoid cells and that the Gal1overexpression by such cells is directly implicated in the developmentand maintenance of a tolerogenic and immunosuppressive microenvironment,leading to an ineffective host anti-proliferative immune response. Thepresent invention is further based, in part, on the discovery thathypoxia promotes upregulation of Gal1 resulting in angiogenesis suchthat targeted disruption of Gal1-glycan lattices attenuates hypoxiaassociated angiogenesis, while promoting pericyte maturation and vacularremoding. Thus, agents such as natural ligands, derivatives of naturalligands, small molecules, RNA interference, aptamer, peptides,peptidomimetics, glycan-related compounds, glycomimetics, and antibodiesthat specifically bind to the Gal1 gene or gene products, or fragmentsthereof, can be utilized for the diagnosis, prognosis, monitoring and/ortreatment of viral-associated PTLD, e.g., EBV-associated PTLD, and/orhypoxia associated angiogenesis disorders. In addition, such agents canbe utilized to modulate, e.g., increase, immune surveillance inviral-associated PTLD, e.g., EBV-associated PTLD and/or downregulatehypoxia associated angiogenesis. Moreover, agents such as Gal1 genesequences, Gal1 gene products, anti-Gal1 RNA interference molecules,anti-Gal1 antibodies (i.e., antibodies that specifically bind to Gal1gene products or fragments thereof), or fragments thereof, can beutilized to restore immune surveillance and neutralization ofviral-associated PTLD, e.g., EBV-associated PTLD, and/or down-regulatehypoxia associated angiogenesis.

Thus, it has been discovered that a higher than normal level ofexpression of Gal1 correlates with the presence of a viral-associatedPTLD, e.g., EBV-associated PTLD, and/or hypoxia associated angiogenesisdisorders in a subject. Gal1 polypeptides and fragments thereof, e.g.,biologically active or antigenic fragments thereof, are provided, asreagents or targets in assays applicable to treatment and/or diagnosisof viral-associated PTLD, e.g., EBV-associated PTLD, and/or hypoxiaassociated angiogenesis disorders. In particular, the methods andcompositions of the present invention relate to detection and/ormodulation of expression and/or activity of a Gal1 gene or fragmentthereof, e.g., biologically active fragments thereof, as well as to thedetection and/or modulation of expression and/or activity of geneproducts or fragments thereof encoded by the Gal1 gene, e.g.,biologically active fragments thereof. The methods of the presentinvention can utilize the Gal1 gene sequence or fragments thereof, aswell as gene products of the Gal1 gene and/or modulators thereof orfragments thereof, e.g., antibodies which specifically bind to such Gal1gene products. The present invention further features methods fordetecting the presence, absence, stage, and other characteristics ofviral-associated PTLD, e.g., EBV-associated PTLD, and/or hypoxiaassociated angiogenesis disorders in a sample that are relevant toprevention, diagnosis, characterization, and therapy in a patient. Inaddition, the present invention also features compositions of matter,including antibodies (e.g., antibodies which specifically bind to anyone of the polypeptides described herein) as well as fusionpolypeptides, including all or a fragment of a polypeptide describedherein. Moreover, the present invention features compositions useful forthe reduction of Gal1 nucleic acids (e.g., Gal1 mRNA or hnRNA orfragments thereof), including RNA interference compositions, directedagainst Gal1 nucleic acids or fragments thereof.

I. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “altered amount” of a marker of a marker refers to increased ordecreased copy number of a marker and/or increased or decreased nucleicacid level of a particular marker gene or genes in a sample, as comparedto that of the marker in a control sample. The term “altered amount” ofa marker also includes an increased or decreased protein level of amarker in a sample, as compared to the protein level of the marker in anormal, control sample.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in abiological sample, as compared to the activity of the marker in anormal, control sample. Altered activity of a marker may be the resultof, for example, altered expression of the marker, altered protein levelof the marker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker, or altered interaction with transcriptionalactivators or inhibitors.

The term “altered structure” of a marker refers to the presence ofmutations or allelic variants within the marker gene or maker protein,e.g., mutations which affect expression or activity of the marker, ascompared to the normal or wild-type gene or protein. For example,mutations include, but are not limited to substitutions, deletions, oraddition mutations. Mutations may be present in the coding or non-codingregion of the marker.

The term “altered subcellular localization” of a marker refers to themislocalization of the marker within a cell relative to the normallocalization within the cell e.g., within a healthy and/or wild-typecell. An indication of normal localization of the marker can bedetermined through an analysis of subcellular localization motifs knownin the field that are harbored by marker polypeptides or, for example,through cellular analyses such as internalization of normallyextracellular mature functional Gal1.

The term “angiogenesis” or “neovascularization” refers to the process bywhich new blood vessels develop from pre-existing vessels [Varner et al.(1999) Angiogen. 3(1):53-60; Mousa et al. (2000) Angiogen. Stim. &Inhib. 35-42; 44. Kim et al. (2000) Amer. J. Path. 156:1345-1362; Kim etal. (2000) J. Biol. Chem. 275:33920-33928; Kumar et al. (2000)Angiogenesis: From Molecular to Integrative Pharm. 169-180]. Endothelialcells from pre-existing blood vessels or from circulating endothelialstem cells [Takahashi et al. (1995) Nat. Med. 5:434-438; Isner et al.(1999) J. Clin. Invest. 103:1231-1236] become activated to migrate,proliferate, and differentiate into structures with lumens, forming newblood vessels, in response to growth factor or hormonal cues, or hypoxicor ischemic conditions. During ischemia, such as occurs in cancer, theneed to increase oxygenation and delivery of nutrients apparentlyinduces the secretion of angiogenic factors by the affected tissue;these factors stimulate new blood vessel formation. Several additionalterms are related to angiogenesis.

For example, the term “tissue exhibiting angiogenesis” refers to atissue in which new blood vessels are developing from pre-existing bloodvessels.

As used herein, the term “inhibiting angiogenesis,” “diminishingangiogenesis,” “reducing angiogenesis,” and grammatical equivalentsthereof refer to reducing the level of angiogenesis in a tissue to aquantity which is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than thequantity in a corresponding control tissue, and most preferably is atthe same level which is observed in a control tissue. A reduced level ofangiogenesis need not, although it may, mean an absolute absence ofangiogenesis. The invention does not require, and is not limited to,methods that wholly eliminate angiogenesis. The level of angiogenesismay be determined using methods well known in the art, including,without limitation, counting the number of blood vessels and/or thenumber of blood vessel branch points, as discussed herein and in theexamples. An alternative in vitro assay contemplated includes thetubular cord formation assay that shows growth of new blood vessels atthe cellular level [D. S. Grant et al., Cell, 58: 933-943 (1989)].Art-accepted in vivo assays are also known, and involve the use ofvarious test animals such as chickens, rats, mice, rabbits and the like.These in vivo assays include the chicken chorioallantoic membrane (CAM)assay, which is suitable for showing anti-angiogenic activity in bothnormal and neoplastic tissues [D. H. Ausprunk, Amer. J. Path., 79, No.3: 597-610 (1975) and L. Ossonowski and E. Reich, Cancer Res., 30:2300-2309 (1980)]. Other in vivo assays include the mouse metastasisassay, which shows the ability of a compound to reduce the rate ofgrowth of transplanted tumors in certain mice, or to inhibit theformation of tumors or preneoplastic cells in mice which are predisposedto cancer or which express chemically-induced cancer [M. J. Humphries etal., Science, 233: 467-470 (1986) and M. J. Humphries et al., J. Clin.Invest., 81: 782-790 (1988)]. Moreover, in some embodiments,angiogenesis can be measured according to such attributes as pericytematuration and vascular remodeling as described further herein.

As used herein, the term “hypoxia associated angiogenesis” or“hypoxia-induced angiogenesis” refers generally to the process ofpathological angiogenesis in non-neoplastic disease states and istypically, although not necessarily, accompanied by a transition to aneoplastic state. Hypoxia-induced transcription factors (HIFs) inducethe expression of angiogeneic factors including HIF-lzlpha, VEGF, nitricoxide synthase, PDFG, Ang2, and others. As a result, hypoxia associatedangiogenesis encompasses a well-known set of pathological conditionscharacterized by such a process Pugh et al. (2003) Nat Med 9, 677-684;Fraisl et al. (2009) Dev Cell 16, 167-179; Ferrara et al. (2005) Nature438, 967-974; Ferrara, N. (2010) Cytokine Growth Factor Rev 21, 21-26].In some embodiments, the set of hypoxia associate angiogenesispathologies includes, but is not limited to, neoplasms and cancers,age-related macular degeneration, diabetes retinopathy, atherosclerosis,chronic obstructive lung disease, and psoriasis.

The term “organized vasculature” means substantially branched bloodvessels, or blood vessels with a normal or increased degree ofbranching, so as to promote blood supply to surrounding tissue. The term“disorganized vasculature” means substantially unbranched blood vessels,or blood vessels with a reduced degree of branching, so as to impairblood supply to surrounding tissue.

Unless otherwise specified here within, the terms “antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies(e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such assingle-chain antibodies, chimeric and humanized antibodies andmulti-specific antibodies, as well as fragments and derivatives of allof the foregoing, which fragments and derivatives have at least anantigenic binding site. Antibody derivatives may comprise a protein orchemical moiety conjugated to an antibody. An “antibody” refers to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. “Inactivating antibodies” refers to antibodies that do notinduce the complement system.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., Gal1 polypeptide or fragment thereof). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent polypeptides (known as single chain Fv (scFv);see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998,Nature Biotechnology 16: 778). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Any VH and VL sequences of specific scFv can be linked tohuman immunoglobulin constant region cDNA or genomic sequences, in orderto generate expression vectors encoding complete IgG polypeptides orother isotypes. VH and VL can also be used in the generation of Fab , Fvor other fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques,as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.).Antibodies may also be fully human. In one embodiment, antibodies of thepresent invention bind specifically or substantially specifically toGal1 polypeptides or fragments thereof. The terms “monoclonalantibodies” and “monoclonal antibody composition”, as used herein, referto a population of antibody polypeptides that contain only one speciesof an antigen binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibodypolypeptides that contain multiple species of antigen binding sitescapable of interacting with a particular antigen. A monoclonal antibodycomposition typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluids that are normally not (e.g. amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum,semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication,vitreous humor, vomit).

The terms “cancer” or “tumor” or “hyperproliferative disorder” refer tothe presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells may exist alone within an animal, or maybe a non-tumorigenic cancer cell, such as a leukemia cell. Cancersinclude, but are not limited to, B cell cancer, e.g., multiple myeloma,Waldenström's macroglobulinemia, the heavy chain diseases, such as, forexample, alpha chain disease, gamma chain disease, and mu chain disease,benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like.

The terms “CDR”, and its plural “CDRs”, refer to a complementaritydetermining region (CDR) of which three make up the binding character ofa light chain variable region (CDRL1, CDRL2 and CDRL3) and three make upthe binding character of a heavy chain variable region (CDRH1, CDRH2 andCDRH3). CDRs contribute to the functional activity of an antibodymolecule and are separated by amino acid sequences that comprisescaffolding or framework regions. The exact definitional CDR boundariesand lengths are subject to different classification and numberingsystems. CDRs may therefore be referred to by Kabat, Chothia, contact orany other boundary definitions. Despite differing boundaries, each ofthese systems has some degree of overlap in what constitutes the socalled “hypervariable regions” within the variable sequences. CDRdefinitions according to these systems may therefore differ in lengthand boundary areas with respect to the adjacent framework region. Seefor example Kabat, Chothia, and/or MacCallum et al., (Kabat et al., in“Sequences of Proteins of Immunological Interest,” 5^(th) Edition, U.S.Department of Health and Human Services, 1992; Chothia et al. (1987) J.Mol. Biol. 196, 901; and MacCallum et al., J. Mol. Biol. (1996) 262,732, each of which is incorporated by reference in its entirety).

As used herein, the term “classifying” includes “to associate” or “tocategorize” a sample with a disease state. In certain instances,“classifying” is based on statistical evidence, empirical evidence, orboth. In certain embodiments, the methods and systems of classifying useof a so-called training set of samples having known disease states. Onceestablished, the training data set serves as a basis, model, or templateagainst which the features of an unknown sample are compared, in orderto classify the unknown disease state of the sample. In certaininstances, classifying the sample is akin to diagnosing the diseasestate of the sample. In certain other instances, classifying the sampleis akin to differentiating the disease state of the sample from anotherdisease state.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. In one embodiment, the first region comprises a firstportion and the second region comprises a second portion, whereby, whenthe first and second portions are arranged in an antiparallel fashion,at least about 50%, and preferably at least about 75%, at least about90%, or at least about 95% of the nucleotide residues of the firstportion are capable of base pairing with nucleotide residues in thesecond portion. In another embodiment, all nucleotide residues of thefirst portion are capable of base pairing with nucleotide residues inthe second portion.

As used herein, the term “composite antibody” refers to an antibodywhich has variable regions comprising germline or non-germlineimmunoglobulin sequences from two or more unrelated variable regions.Additionally, the term “composite, human antibody” refers to an antibodywhich has constant regions derived from human germline or non-germlineimmunoglobulin sequences and variable regions comprising human germlineor non-germline sequences from two or more unrelated human variableregions. A composite, human antibody is useful as an effective componentin a therapeutic agent according to the present invention since theantigenicity of the composite, human antibody in the human body islowered.

As used herein, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain, including native-sequence Fcregions and variant Fc regions. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy-chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof.Suitable native-sequence Fc regions for use in the antibodies of thepresent invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 andIgG4.

As used herein, “Fc receptor” or “FcR” describes a receptor that bindsto the Fc region of an antibody. The preferred FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one which binds an IgG antibody(a gamma receptor) and includes receptors of the Fc RI, Fc RII, and FcRII subclasses, including allelic variants and alternatively splicedforms of these receptors, Fc RII receptors include Fc RIIA (an“activating receptor”) and Fc RIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor Fc RIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor Fc RIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M.Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al.,Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

A molecule is “fixed” or “affixed” to a substrate if it is covalently ornon-covalently associated with the substrate such the substrate can berinsed with a fluid (e.g. standard saline citrate, pH 7.4) without asubstantial fraction of the molecule dissociating from the substrate.

As used herein, “Framework” or “FR” residues are those variable-domainresidues other than the HVR residues as herein defined.

As used herein, the term “heterologous antibody” is defined in relationto the transgenic non-human organism producing such an antibody. Thisterm refers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the present invention, such as a recombinantexpression vector of the present invention, has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It should be understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the presentinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs. The term “humanized antibody”, as used herein, alsoincludes antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

As used herein, the term “hypervariable region,” “HVR,” or “HV,” refersto the regions of an antibody-variable domain that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.(2000) Immunity 13, 37-45; Johnson and Wu in Methods in MolecularBiology 248, 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003)). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain (see, e.g.,Hamers-Casterman et al. (1993) Nature 363:446-448 (1993) and Sheriff etal. (1996) Nature Struct. Biol. 3, 733-736).

As used herein, the term “immune cell” refers to cells that play a rolein the immune response. Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “immune disorder” includes immune diseases,conditions, and predispositions to, including, but not limited to,Hodgkin lymphoma (including, e.g., lymphocyte-rich classical Hodgkinlymphoma, mixed cellularity classical Hodgkin lymphoma,lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosisclassical Hodgkin lymphoma, anaplastic large cell lymphoma, or MLL preB-cell ALL), cancer, chronic inflammatory disease and disorders(including, e.g., Crohn's disease, inflammatory bowel disease, reactivearthritis, and Lyme disease), insulin-dependent diabetes, organ specificautoimmunity (including, e.g., multiple sclerosis, Hashimoto'sthyroiditis, autoimmune uveitis, and Grave's disease), contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions (including, e.g., asthma and allergyincluding, but not limited to, allergic rhinitis and gastrointestinalallergies such as food allergies), eosinophilia, conjunctivitis,glomerular nephritis, systemic lupus erythematosus, scleroderma, certainpathogen susceptibilities such as helminthic (including, e.g.,leishmaniasis) and certain viral infections (including, e.g., HIV andbacterial infections such as tuberculosis and lepromatous leprosy).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production, and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages.

As used herein, the term “inhibit” includes the decrease, limitation, orblockage, of, for example a particular action, function, or interaction.

As used herein, the term “interaction”, when referring to an interactionbetween two molecules, refers to the physical contact (e.g., binding) ofthe molecules with one another. Generally, such an interaction resultsin an activity (which produces a biological effect) of one or both ofsaid molecules. The activity may be a direct activity of one or both ofthe molecules, (e.g., signal transduction). Alternatively, one or bothmolecules in the interaction may be prevented from binding their ligand,and thus be held inactive with respect to ligand binding activity (e.g.,binding its ligand and triggering or inhibiting an immune response). Toinhibit such an interaction results in the disruption of the activity ofone or more molecules involved in the interaction. To enhance such aninteraction is to prolong or increase the likelihood of said physicalcontact, and prolong or increase the likelihood of said activity.

As used herein, an “antisense” nucleic acid polypeptide comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA polypeptide, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid polypeptide can hydrogen bond to a sense nucleic acidpolypeptide.

As used herein, the term an “isolated antibody” is intended to refer toan antibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to human Gal1 and is substantially free of antibodiesthat do not bind to Gal1). An isolated antibody that specifically bindsto an epitope of human Gal1 may, however, have cross-reactivity to otherGal1 proteins, respectively, from different species. However, in someembodiments, the antibody maintains higher affinity and selectivity forhuman Gal1. In addition, an isolated antibody is typically substantiallyfree of other cellular material and/or chemicals. In one embodiment ofthe present invention, a combination of “isolated” monoclonal antibodieshaving different specificities to human Gal1 are combined in a welldefined composition.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material, separationmedium, and culture medium when isolated from cells or produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. An “isolated” or “purified” protein orbiologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the antibody, polypeptide, peptide or fusion protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of Gal1 polypeptide orfragment thereof, in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of Gal1 protein or fragmentthereof, having less than about 30% (by dry weight) of non-Gal1 protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-Gal1 protein, still more preferably less thanabout 10% of non-Gal1 protein, and most preferably less than about 5%non-Gal1 protein. When antibody, polypeptide, peptide or fusion proteinor fragment thereof, e.g., a biologically active fragment thereof, isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

As used herein, the term “isotype” refers to the antibody class (e.g.,IgM or IgG1) that is encoded by heavy chain constant region genes.

As used herein, the term “K_(D)” is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction. The binding affinity of antibodies of the disclosedinvention may be measured or determined by standard antibody-antigenassays, for example, competitive assays, saturation assays, or standardimmunoassays such as ELISA or RIA.

As used herein, a “kit” is any manufacture (e.g. a package or container)comprising at least one reagent, e.g. a probe, for specificallydetecting or modulating the expression of a marker of the presentinvention. The kit may be promoted, distributed, or sold as a unit forperforming the methods of the present invention.

As used herein, the term “monoclonal antibody”, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and constant regions derived from human germline ornon-germline immunoglobulin sequences. In one embodiment, humanmonoclonal antibodies are produced by a hybridoma which includes a Bcell obtained from a transgenic non-human animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

A “post-transplantation lymphoproliferative disorder”, “PTLD”, and/or“viral-associated PTLD” each refers to a disorder in which lymphocytes,which are white blood cells produced in the lymphatic tissue (e.g.,lymph nodes, spleen, and/or thymus), are over-produced or act abnormallyand are caused by or correlated with a virus. Lymphoid cells includethymus derived lymphocytes (T cells); bone marrow-derived lymphocytes (Bcells), and natural killer (NK cells), for example. Lymphocytes progressthrough a number of different stages, including proliferation,activation, and maturation, and lymphoma or aberrant proliferation candevelop at each stage. Disorders may be malignant neoplasms (and may beclassified as aggressive or indolent, or as low, intermediate orhigh-grade), including those associated with IFN-.gamma., or thedisorders may involve non-malignant aberrant expansion of lymphoidcells. LPDs include any monoclonal or polyclonal LPD that is notresolving without treatment and/or that involves excessive cellularproliferation, such as an expanding, monoclonal, polyclonal oroligoclonal, lymphoid neoplasm. Cellular proliferation may be more rapidthan normal and may continue after the stimuli that initiated the newgrowth cease. A neoplasm will show partial or complete lack ofstructural organization and functional coordination with the normaltissue, and may form a distinct mass of tissue that may be either benign(benign tumor) or malignant (cancer).

Such viral-associated PTLD may be caused by or associated with, e.g.,Epstein-Barr virus (EBV), a herpes virus, HHV-8, cytomegalovirus, C-typeretrovirus, human T-lymphotropic virus type 1 (C-type retrovirus),and/or human immunodeficiency virus (HIV, HIV-1, HIV-2). HIV- and/orAIDS-associated cancers include HIV-associated LPDs, such as Karposisarcoma, non-Hodgkin's lymphoma, central nervous system (CNS) lymphoma,adult T-cell leukemia/lymphoma (HTLV-1+), and AIDS-associated lymphoma.Immune deficiency such as in AIDS patients, organ transplant recipients,and genetic immune disorders may allow latent EBV to reactivate, causingproliferation of abnormal lymphocytes and the potential to develop anEBV-associated LPD, for example. Methods to detect the presence of virusor viral infection in an aberrant cell, such as a cell involved in aPTLD, are known in the art. Viral nucleic acids or polypeptides may bedetected in a cell, tissue, or organism such as an aberrant cell, forexample. Also, methods to detect immune response specific for a virusare known. A delayed type-hypersensitivity (DTH) assay, such as a transvivo DTH assay may be used to detect regulatory T cells, for example. Insuch an assay, human or other mammalian peripheral blood mononuclearcells (PBMC) may be mixed with a carrier control with and without viralantigen, for example, and injected into a heterologous naive recipient,such as the pinnae or footpad of naive mice. If the donor of the PBMChad previously been sensitized to the challenge antigen, DTH-likeswelling responses are observed.

A “marker” is a gene whose altered level of expression in a tissue orcell from its expression level in normal or healthy tissue or cell isassociated with a disease state, such as cancer. A “marker nucleic acid”is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to amarker of the present invention. Such marker nucleic acids include DNA(e.g., cDNA) comprising the entire or a partial sequence of any of thenucleic acid sequences set forth in the Sequence Listing or thecomplement of such a sequence. The marker nucleic acids also include RNAcomprising the entire or a partial sequence of any of the nucleic acidsequences set forth in the Sequence Listing or the complement of such asequence, wherein all thymidine residues are replaced with uridineresidues. A “marker protein” is a protein encoded by or corresponding toa marker of the present invention. A marker protein comprises the entireor a partial sequence of any of the sequences set forth in the SequenceListing. The terms “protein” and “polypeptide” are used interchangeably.

As used herein, the term “modulate” includes up-regulation anddown-regulation, e.g., enhancing or inhibiting a response.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject, e.g., a human patient, notafflicted with a viral-associated PTLD. An “over-expression” or“significantly higher level of expression” of a marker refers to anexpression level in a test sample that is greater than the standarderror of the assay employed to assess expression, and is preferably atleast twice, and more preferably three, four, five or ten times theexpression level of the marker in a control sample (e.g., sample from ahealthy subjects not having the marker associated disease) andpreferably, the average expression level of the marker in severalcontrol samples. A “significantly lower level of expression” of a markerrefers to an expression level in a test sample that is at least twice,and more preferably three, four, five or ten times lower than theexpression level of the marker in a control sample (e.g., sample from ahealthy subject not having the marker associated disease) andpreferably, the average expression level of the marker in severalcontrol samples.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA. As used herein, the term “isolated nucleic acid molecule” inreference to nucleic acids encoding antibodies or antibody portions(e.g., V_(H), V_(L), CDR3) that bind to Gal1, is intended to refer to anucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thanGal1, which other sequences may naturally flank the nucleic acid inhuman genomic DNA.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

An “over-expression” or “significantly higher level of expression” of amarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andis preferably at least twice, and more preferably 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or morehigher than the expression activity or level of the marker in a controlsample (e.g., sample from a healthy subject not having the markerassociated disease) and preferably, the average expression level of themarker in several control samples. A “significantly lower level ofexpression” of a marker refers to an expression level in a test samplethat is at least twice, and more preferably 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or morelower than the expression level of the marker in a control sample (e.g.,sample from a healthy subject not having the marker associated disease)and preferably, the average expression level of the marker in severalcontrol samples.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions mayoccur at the amino-terminus, internally, or at the carboxy-terminus ofthe reference polypeptide, or alternatively both. Fragments typicallyare at least 5, 6, 8 or 10 amino acids long, at least 14 amino acidslong, at least 20, 30, 40 or 50 amino acids long, at least 75 aminoacids long, or at least 100, 150, 200, 300, 500 or more amino acidslong. They can be, for example, at least and/or including 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120,140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680,700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960,980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160, 1180, 1200,1220, 1240, 1260, 1280, 1300, 1320, 1340 or more long so long as theyare less than the length of the full-length polypeptide. Alternatively,they can be no longer than and/or excluding such a range so long as theyare less than the length of the full-length polypeptide. The VEGFR2-GAL1intereaction involves N-glycosylation sites as it is prevented bytreatment with swainsoninc or siRNA-mediated silencing of GnT5glycosyltransferasem, which is responsible for generating complexN-glycans. A fragment can retain one or more of the biologicalactivities of the reference polypeptide. In various embodiments, afragment may comprise an enzymatic activity and/or an interaction siteof the reference polypeptide. In another embodiment, a fragment may haveimmunogenic properties. In an exemplary embodiment the fragmentcomprises a binding domain. In one exemplary embodiment a Gal1 fragmentis able to form a complex with a VEGFR2 polypeptide, or a fragmentthereof. In another embodiment a VEGFR2 fragment is able to form acomplex with a Gal1 polypeptide, or a fragment thereof.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example, anucleotide transcript or protein encoded by or corresponding to amarker. Probes can be either synthesized by one skilled in the art, orderived from appropriate biological preparations. For purposes ofdetection of the target molecule, probes may be specifically designed tobe labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

As used herein, the term “rearranged” refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) and V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

As used herein, the term “recombinant host cell” (or simply “hostcell”), is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “recombinant human antibody” includes all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express theantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regionsderived from human germline and/or non-germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

The present invention “response” is generally related to for example,determining the effects on progression, efficacy, or outcome of aclinical intervention. In some embodiments, responses relate directly toa change in tumor mass and/or volume after initiation of clinicalintervention (e.g., administration of an anti-Gal1 monoclonal antibody).For example, hyperproliferative disorder responses may be assessedaccording to the size of a tumor after systemic intervention compared tothe initial size and dimensions as measured by CT, PET, mammogram,ultrasound or palpation. Response may also be assessed by calipermeasurement or pathological examination of the tumor after biopsy orsurgical resection. Response may be recorded in a quantitative fashionlike percentage change in tumor volume or in a qualitative fashion like“pathological complete response” (pCR), “clinical complete remission”(cCR), “clinical partial remission” (cPR), “clinical stable disease”(cSD), “clinical progressive disease” (cPD) or other qualitativecriteria. Assessment may be done early after the onset of the clinicalintervention, e.g., after a few hours, days, weeks or preferably after afew months. A typical endpoint for response assessment is upontermination of the clinical intervention or upon surgical removal ofresidual tumor cells and/or the tumor bed.

As used herein, the term “specific binding” refers to antibody bindingto a predetermined antigen. Typically, the antibody binds with anaffinity (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE®assay instrument using human FAS and/or USP2a as the analyte and theantibody as the ligand, and binds to the predetermined antigen with anaffinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-,1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-,9.0-, or 10.0-fold or greater than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen. The phrases “an antibodyrecognizing an antigen” and “an antibody specific for an antigen” areused interchangeably herein with the term “an antibody which bindsspecifically to an antigen.”

As used herein, “subject” refers to any healthy animal, mammal or human,or any animal, mammal or human afflicted with a viral-associated PTLD,e.g., EBV-associated PTLD. The term “subject” is interchangeable with“patient”. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of antibody, polypeptide, peptide orfusion protein in which the protein is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of antibody,polypeptide, peptide or fusion protein having less than about 30% (bydry weight) of chemical precursors or non-antibody, polypeptide, peptideor fusion protein chemicals, more preferably less than about 20%chemical precursors or non-antibody, polypeptide, peptide or fusionprotein chemicals, still more preferably less than about 10% chemicalprecursors or non-antibody, polypeptide, peptide or fusion proteinchemicals, and most preferably less than about 5% chemical precursors ornon-antibody, polypeptide, peptide or fusion protein chemicals.

As used herein, the term “survival” includes all of the following:survival until mortality, also known as overall survival (wherein saidmortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall includeboth localized and distant recurrence); metastasis free survival;disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g. time of diagnosisor start of treatment) and end point (e.g. death, recurrence ormetastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the present inventionand normal post-transcriptional processing (e.g. splicing), if any, ofthe RNA transcript, and reverse transcription of the RNA transcript.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells. The term “antigen presenting cell” includesprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, Langerhans cells) as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

As used herein, the term “unrearranged” or “germline configuration” inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

As used herein, the term “vector” refers to a nucleic acid capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” or simply “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

Genetic Code

Alanine  GCA, GCC, GCG, GCT (Ala, A) Arginine AGA, ACG, CGA, CGC, CGG, CGT (Arg, R) Asparagine  AAC, AAT (Asn, N)Aspartic acid  GAC, GAT (Asp, D) Cysteine  TGC, TGT (Cys, C)Glutamic acid  GAA, GAG (Glu, E) Glutamine  CAA, CAG (Gln, Q) Glycine GGA, GGC, GGG, GGT (Gly, G) Histidine  CAC, CAT (His, H) Isoleucine ATA, ATC, ATT (Ile, I) Leucine  CTA, CTC, CTG, CTT, TTA, TTG (Leu, L)Lysinc  AAA, AAG (Lys, K) Methionine  ATG (Met, M) Phenylalanine TTC, TTT (Phe, F) Proline  CCA, CCC, CCG, CCT (Pro, P) Serine AGC, AGT, TCA, TCC, TCG, TCT (Ser, S) Threonine  ACA, ACC, ACG, ACT(Thr, T) Tryptophan  TGG (Trp, W) Tyrosine  TAC, TAT (Tyr, Y) Valine GTA, GTC, GTG, GTT (Val, V) Termination signal  TAA, TAG, TGA (end)

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, or more of the nucleotides, and more preferably at leastabout 97%, 98%, 99% or more of the nucleotides. Alternatively,substantial homology exists when the segments will hybridize underselective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available on theworld wide web at the GCG company website), using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6. The percent identity between two nucleotide or amino acidsequences can also be determined using the algorithm of E. Meyers and W.Miller (CABIOS, 4:11 17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm whichhas been incorporated into the GAP program in the GCG software package(available on the world wide web at the GCG company website), usingeither a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403 10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to the protein molecules of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389 3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used (available on the world wide web at theNCBI website).

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art (see, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987)).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

In view of the foregoing, the nucleotide sequence of a DNA or RNA codingfor a fusion protein or polypeptide of the present invention (or anyportion thereof) can be used to derive the fusion protein or polypeptideamino acid sequence, using the genetic code to translate the DNA or RNAinto an amino acid sequence. Likewise, for fusion protein or polypeptideamino acid sequence, corresponding nucleotide sequences that can encodethe fusion protein or polypeptide can be deduced from the genetic code(which, because of its redundancy, will produce multiple nucleic acidsequences for any given amino acid sequence). Thus, description and/ordisclosure herein of a nucleotide sequence which encodes a fusionprotein or polypeptide should be considered to also include descriptionand/or disclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a fusion proteinor polypeptide amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

II. Description

The present invention relates, in part, to compositions, kits, andmethods for the diagnosis, prognosis, monitoring, and modulation ofviral-associated PTLD and/or hypoxia associated angiogenesis of a genereferred to herein as the galectin-1 (Gal1) gene or a fragment thereof.In particular, the methods and compositions of the present inventionrelate to detection and/or modulation of expression and/or activity ofGal1 or a fragment thereof, e.g., a biologically active fragmentthereof, as well as to the detection and/or modulation of expressionand/or activity of gene products encoded by the Gal1 gene (i.e., a “Gal1gene product”) or fragments thereof, e.g., biologically active fragmentsthereof. The present invention can utilize the Gal1 gene sequence orfragments thereof, as well as gene products of the Gal1 gene and/ormodulators thereof, e.g., antibodies which specifically bind to suchGal1 gene products, or fragments thereof.

Sequences, structures, domains, biophysical characteristics, andfunctions of Gal1 gene and gene products have been described in the art.See, for example, Rabinovich et al. (2002) Trends Immunol 23:313-320;Liu and Rabinovich (2005) Nature Reviews Cancer 5:29-41; Rubinstein etal. (2004) Cancer Cell 5:241-251; Le et al. (2005) J Clin Oncol23:8932-8941; Vasta et al. (2004) Curr Opin Struct Biol 14:617-630;Toscano et al. (2007) Cyt Growth Fact Rev 18:57-71; Camby et al. (2006)Glycobiol 16:137R-157R, each of which is incorporated herein, byreference, in its entirety. Gal1 gene and gene products from manyspecies are known and include, for example, chimpanzee Gal1 (NCBIAccession XM_(—)001162066), rat Gal1 (NCBI Accession NM_(—)019904),mouse Gal1 (NM_(—)008495), and chicken Gal1 (NM_(—)205495). Human Gal1sequences include those listed below.

Gal1 coding nucleic acid sequence (SEQ ID NO: 1):

ATGGCTTGTG GTCTGGTCGC CAGCAACCTG AATCTCAAACCTGGAGAGTG CCTTCGAGTG CGAGGCGAGG TGGCTCCTGACGCTAAGAGC TTCGTGCTGA ACCTGGGCAA AGACAGCAACAACCTGTGCC TGCACTTCAA CCCTCGCTTC AACGCCCACGGCGACGCCAA CACCATCGTG TGCAACAGCA AGGACGGCGGGGCCTGGGGG ACCGAGCAGC GGGAGGCTGT CTTTCCCTTCCAGCCTGGAA GTGTTGCAGA GGTGTGCATC ACCTTCGACCAGGCCAACCT GACCGTCAAG CTGCCAGATG GATACGAATTCAAGTTCCCC AACCGCCTCA ACCTGGAGGC CATCAACTACATGGCAGCTG ACGGTGACTT CAAGATCAAA TGTGTGGCCT TTGACTGA

Gal1 protein sequence (SEQ ID NO: 2):

MACGLVASNL NLKPGECLRV RGEVAPDAKS FVLNLGKDSNNLCLHFNPRF NAHGDANTIV CNSKDGGAWG TEQREAVFPFQPGSVAEVCI TFDQANLTVK LPDGYEFKFP NRLNLEAINY MAADGDFKIK CVAFD

Similarly, sequences, structures, domains, biophysical characteristics,and functions of VEGFR2 gene and gene products, and glycosylated formsthereof, have been described in the art. See, for example, Terman et al.(1992) Biochem. Biophys. Res. Commun. 187:1579-1586; Witte et al. (1998)Cancer Metastasis 17:155-161; Ortega et al. (1999) Front. Biosci.4:D141-D152; Shibuya (2002) Biol. Chem. 383:1573-1579; Olsson et al.(2006) Nat. Rev. Mol. Cell. Biol. 7:359-371; and Shibuya (2006) J.Biochem. Mol. Biol. 39:469-478; each of which is incorporated herein, byreference, in its entirety. VEGFR2 gene and gene products from manyspecies are known and include, for example, chimpanzee VEGFR2 (NCBIAccession XM_(—)517284.2 and XP_(—)517284.2), dog VEGFR2 (NCBI AccessionXM_(—)539273.2 and XP_(—)539273.2), cow VEGFR2 (NCBI AccessionXM_(—)611785.3 and XP_(—)611785.3), mouse VEGFR2 (NCBI AccessionNM_(—)010612.2 and NP_(—)034742.2) and chicken Gal1 (NM_(—)001004368.1and NP_(—)001004368.1). Human VEGFR2 sequences include those listedbelow. In addition, glycosylated forms of VEGFR2 all known in the art asdescribed, for example, by Zhang et al. (2010) Cell Death Differ.17:499, which is incorporated herein, by reference, in its entirety.

VEGFR2 coding nucleic acid sequence (NM_(—)002253.2) (SEQ ID NO: 3):

   1 atgcagagca aggtgctgct ggccgtcgcc ctgtggctct gcgtggagac ccgggccgcc  61 tctgtgggtt tgcctagtgt ttctcttgat ctgcccaggc tcagcataca aaaagacata 121 cttacaatta aggctaatac aactcttcaa attacttgca ggggacagag ggacttggac 181 tggctttggc ccaataatca gagtggcagt gagcaaaggg tggaggtgac tgagtgcagc 241 gatggcctct tctgtaagac actcacaatt ccaaaagtga tcggaaatga cactggagcc 301 tacaagtgct tctaccggga aactgacttg gcctcggtca tttatgtcta tgttcaagat 361 tacagatctc catttattgc ttctgttagt gaccaacatg gagtcgtgta cattactgag 421 aacaaaaaca aaactgtggt gattccatgt ctcgggtcca tttcaaatct caacgtgtca 481 ctttgtgcaa gatacccaga aaagagattt gttcctgatg gtaacagaat ttcctgggac 541 agcaagaagg gctttactat tcccagctac atgatcagct atgctggcat ggtcttctgt 601 gaagcaaaaa ttaatgatga aagttaccag tctattatgt acatagttgt cgttgtaggg 661 tataggattt atgatgtggt tctgagtccg tctcatggaa ttgaactatc tgttggagaa 721 aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg 781 gaataccctt cttcgaagca tcagcataag aaacttgtaa accgagacct aaaaacccag 841 tctgggagtg agatgaagaa atttttgagc accttaacta tagatggtgt aacccggagt 901 gaccaaggat tgtacacctg tgcagcatcc agtgggctga tgaccaagaa gaacagcaca 961 tttgtcaggg tccatgaaaa accttttgtt gcttttggaa gtggcatgga atctctggtg1021 gaagccacgg tgggggagcg tgtcagaatc cctgcgaagt accttggtta cccaccccca1081 gaaataaaat ggtataaaaa tggaataccc cttgagtcca atcacacaat taaagcgggg1141 catgtactga cgattatgga agtgagtgaa agagacacag gaaattacac tgtcatcctt1201 accaatccca tttcaaagga gaagcagagc catgtggtct ctctggttgt gtatgtccca1261 ccccagattg gtgagaaatc tctaatctct cctgtggatt cctaccagta cggcaccact1321 caaacgctga catgtacggt ctatgccatt cctcccccgc atcacatcca ctggtattgg1381 cagttggagg aagagtgcgc caacgagccc agccaagctg tctcagtgac aaacccatac1441 ccttgtgaag aatggagaag tgtggaggac ttccagggag gaaataaaat tgaagttaat1501 aaaaatcaat ttgctctaat tgaaggaaaa aacaaaactg taagtaccct tgttatccaa1561 gcggcaaatg tgtcagcttt gtacaaatgt gaagcggtca acaaagtcgg gagaggagag1621 agggtgatct ccttccacgt gaccaggggt cctgaaatta ctttgcaacc tgacatgcag1681 cccactgagc aggagagcgt gtctttgtgg tgcactgcag acagatctac gtttgagaac1741 ctcacatggt acaagcttgg cccacagcct ctgccaatcc atgtgggaga gttgcccaca1801 cctgtttgca agaacttgga tactctttgg aaattgaatg ccaccatgtt ctctaatagc1861 acaaatgaca ttttgatcat ggagcttaag aatgcatcct tgcaggacca aggagactat1921 gtctgccttg ctcaagacag gaagaccaag aaaagacatt gcgtggtcag gcagctcaca1981 gtcctagagc gtgtggcacc cacgatcaca ggaaacctgg agaatcagac gacaagtatt2041 ggggaaagca tcgaagtctc atgcacggca tctgggaatc cccctccaca gatcatgtgg2101 tttaaagata atgagaccct tgtagaagac tcaggcattg tattgaagga tgggaaccgg2161 aacctcacta tccgcagagt gaggaaggag gacgaaggcc tctacacctg ccaggcatgc2221 agtgttcttg gctgtgcaaa agtggaggca tttttcataa tagaaggtgc ccaggaaaag2281 acgaacttgg aaatcattat tctagtaggc acggcggtga ttgccatgtt cttctggcta2341 cttcttgtca tcatcctacg gaccgttaag cgggccaatg gaggggaact gaagacaggc2401 tacttgtcca tcgtcatgga tccagatgaa ctcccattgg atgaacattg tgaacgactg2461 ccttatgatg ccagcaaatg ggaattcccc agagaccggc tgaagctagg taagcctctt2521 ggccgtggtg cctttggcca agtgattgaa gcagatgcct ttggaattga caagacagca2581 acttgcagga cagtagcagt caaaatgttg aaagaaggag caacacacag tgagcatcga2641 gctctcatgt ctgaactcaa gatcctcatt catattggtc accatctcaa tgtggtcaac2701 cttctaggtg cctgtaccaa gccaggaggg ccactcatgg tgattgtgga attctgcaaa2761 tttggaaacc tgtccactta cctgaggagc aagagaaatg aatttgtccc ctacaagacc2821 aaaggggcac gattccgtca agggaaagac tacgttggag caatccctgt ggatctgaaa2881 cggcgcttgg acagcatcac cagtagccag agctcagcca gctctggatt tgtggaggag2941 aagtccctca gtgatgtaga agaagaggaa gctcctgaag atctgtataa ggacttcctg3001 accttggagc atctcatctg ttacagcttc caagtggcta agggcatgga gttcttggca3061 tcgcgaaagt gtatccacag ggacctggcg gcacgaaata tcctcttatc ggagaagaac3121 gtggttaaaa tctgtgactt tggcttggcc cgggatattt ataaagatcc agattatgtc3181 agaaaaggag atgctcgcct ccctttgaaa tggatggccc cagaaacaat ttttgacaga3241 gtgtacacaa tccagagtga cgtctggtct tttggtgttt tgctgtggga aatattttcc3301 ttaggtgctt ctccatatcc tggggtaaag attgatgaag aattttgtag gcgattgaaa3361 gaaggaacta gaatgagggc ccctgattat actacaccag aaatgtacca gaccatgctg3421 gactgctggc acggggagcc cagtcagaga cccacgtttt cagagttggt ggaacatttg3481 ggaaatctct tgcaagctaa tgctcagcag gatggcaaag actacattgt tcttccgata3541 tcagagactt tgagcatgga agaggattct ggactctctc tgcctacctc acctgtttcc3601 tgtatggagg aggaggaagt atgtgacccc aaattccatt atgacaacac agcaggaatc3661 agtcagtatc tgcagaacag taagcgaaag agccggcctg tgagtgtaaa aacatttgaa3721 gatatcccgt tagaagaacc agaagtaaaa gtaatcccag atgacaacca gacggacagt3781 ggtatggttc ttgcctcaga agagctgaaa actttggaag acagaaccaa attatctcca3841 tcttttggtg gaatggtgcc cagcaaaagc agggagtctg tggcatctga aggctcaaac3901 cagacaagcg gctaccagtc cggatatcac tccgatgaca cagacaccac cgtgtactcc3961 agtgaggaag cagaactttt aaagctgata gagattggag tgcaaaccgg tagcacagcc4021 cagattctcc agcctgactc ggggaccaca ctgagctctc ctcctgttta a

VEGFR2 protein sequence (NP_(—)002244.1) (SEQ ID NO: 4):

   1 mqskvllava lwlcvetraa svglpsysld lprlsiqkdi ltikanttlq itcrgqrdld  61 wlwpnnqsgs eqrvevtecs dglfcktlti pkvigndtga ykcfyretdl asviyvyvqd 121 yrspfiasys dqhgvvyite nknktvvipc lgsisnlnvs lcarypekrf vpdgnriswd 181 skkgftipsy misyagmvfc eakindesyq simyivvvvg yriydvvlsp shgielsvge 241 klvlnctart elnvgidfnw eypsskhqhk klvnrdlktq sgsemkkfls tltidgvtrs 301 dqglytcaas sglmtkknst fvrvhekpfv afgsgmeslv eatvgervri pakylgyppp 361 eikwykngip lesnhtikag hvltimevse rdtgnytvil tnpiskekqs hvvslvvyvp 421 pqigekslis pvdsyqygtt qtltctvyai ppphhihwyw qleeecanep sqavsvtnpy 481 pceewrsved fqggnkievn knqfaliegk nktvstlviq aanvsalykc eavnkvgrge 541 rvisfhvtrg peitlqpdmq pteqesyslw ctadrstfen ltwyklgpqp lpihvgelpt 601 pvcknldtlw klnatmfsns tndilimelk naslqdqgdy vclaqdrktk krhcvvrqlt 661 vlervaptit gnlenqttsi gesievscta sgnpppqimw fkdnetlved sgivlkdgnr 721 nltirrvrke deglytcqac svlgcakvea ffiiegaqek tnleiiilvg taviamffwl 781 llviilrtvk ranggelktg ylsivmdpde lpldehcerl pydaskwefp rdrlklgkpl 841 grgafgqvie adafgidkta tcrtvavkml kegathsehr almselkili highhlnvvn 901 llgactkpgg plmvivefck fgnlstylrs krnefvpykt kgarfrqgkd yvgaipvdlk 961 rrldsitssq ssassgfvee kslsdveeee apedlykdfl tlehlicysf qvakgmefla1021 srkcihrdla arnillsekn vvkicdfgla rdiykdpdyv rkgdarlplk wmapetifdr1081 vytiqsdvws fgvllweifs lgaspypgvk ideefcrrlk egtrmrapdy ttpemyqtml1141 dcwhgepsqr ptfselvehl gnllqanaqq dgkdyivlpi setlsmeeds glslptspvs1201 cmeeeevcdp kfhydntagi sqylqnskrk srpvsvktfe dipleepevk vipddnqtds1261 gmvlaseelk tledrtklsp sfggmvpsks resvasegsn qtsgyqsgyh sddtdttvys1321 seeaellkli eigvqtgsta qilqpdsgtt lssppv

The present invention is based, in part, on the discovery that Gal1 isoverexpressed by viral-associated post-transplantation lymphoblastoidcells and that the Gal1 overexpression by such cells is directlyimplicated in the development and maintenance of a tolerogenic andimmunosuppressive microenvironment, leading to an ineffective hostanti-proliferative immune response. The present invention is furtherbased, in part, on the discovery that hypoxia promotes upregulation ofGal1, which results in angiogenesis mediated by VEGFR2 signaling andwhose targeted disruption downregulates hypoxia-driven angiogenesis,while promoint pericyte maturation and vascular remodeling, Thus, agentssuch as natural ligands, derivatives of natural ligands, and smallmolecules, RNA interference, aptamer, peptides, peptidomimetics,glycan-related compounds, glycomimetics, and antibodies thatspecifically bind to the Gal1 gene or gene products or fragments thereofcan be utilized to modulate (e.g., increase) immune surveillance inviral-associated PTLD, e.g., EBV-associated PTLD, and/or hypoxiaassociated angiogenesis disorders. Additionally, agents such as Gal1gene sequences, Gal1 gene products, anti-Gal1 RNA interferencemolecules, anti-Gal1 antibodies (i.e., antibodies that specifically bindto Gal1 gene products), or fragments thereof, can be utilized to reducethe level of TH2 cell activity and/or increase the level of TH1 cellactivity to restore immune surveillance in viral-associated PTLD, e.g.,EBV-associated PTLD, and/or down-regulate hypoxia associatedangiogenesis associated.

The Gal1 gene is also expressed in other cells known in the art. See,for example, Gottschalk et al. (2005) Annu. Rev. Med. 56, 29-44;Nalesnik et al. (1999) Clin. Transplant. 13, 39-44; Toscano et al.(2007) Nat. Immunol. 8, 825-834; Ilarregui et al. (2009) Nat. Immunol.10, 981-991; Re et al. (2005) J. Clin. Oncol. 23, 6379-6386; Marshall etal. (2004) Blood 103, 1755-1762; Gandhi et al. (2006) Blood 108,2280-2289; Juszczynski et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104,13134-13139; Rodig et al. (2008) Clin. Cancer Res. 14, 3338-3344;Rabinovich et al. (2002) Trends Immunol 23:313-320; Liu and Rabinovich(2005) Nature Reviews Cancer 5:29-41; Rubinstein et al. (2004) CancerCell 5:241-251; Le et al. (2005) J Clin Oncol 23:8932-8941; Vasta et al.(2004)Curr Opin Struct Biol 14:617-630; Toscano et al. (2007) Cyt GrowthFact Rev 18:57-71; Camby et al. (2006) Glycobiol 16:137 R-157R, each ofwhich is incorporated herein, by reference, in its entirety. Thus, theabove-described compositions (e.g., natural ligands, derivatives ofnatural ligands, and small molecules, RNA interference, aptamer,peptides, peptidomimetics, glycan-related compounds, glycomimetics,antibodies that specifically bind to the Gal1 gene or gene products, orfragments thereof) can also be utilized to modulate immune responses inthese immune-related cells.

III. Agents that Modulate Immune Cell Activation

The agents of the invention can modulate, e.g., up or down regulate,expression and/or activity of gene products or fragments thereof encodedby the Gal1 gene or fragment thereof and, thereby, modulate, e.g., up ordownregulate, an immune response. The interaction between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof in the context of cHL results in a tolerogenicand/or immunosuppressive microenvironment. Thus, in one embodiment,agents which block the interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof can enhance an immune response (e.g., restore immunesurveillance in viral-associated PTLD, e.g., EBV-associated PTLD),and/or down-regulate hypoxia associated angiogenesis. In anotherembodiment, agents that increase the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof can decrease an immune response (e.g.,immunosuppression). Exemplary agents for modulating a Gal1-mediatedimmune response include antibodies against Gal1 which inhibit theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof; small molecules,peptides, peptidomimetics, glycan-related compounds, glycomimetics,natural ligands, and derivatives of natural ligands, which inhibit theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof; and RNAinterference, antisense, and nucleic acid aptamers that reduce Gal1nucleic acids or Gal1 expression products or fragments thereof.

1. Isolated Nucleic Acid Molecules

One aspect of the present invention pertains to isolated nucleic acidmolecules that encode polypeptides of the present invention (e.g.,including the sequences in Table 1) or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify nucleic acid molecules encoding thesepolypeptides and fragments for use as PCR primers for the amplificationor mutation of the nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. In some embodiments an “isolated” nucleicacid molecule is free of sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acidmolecule) in the genomic DNA of the organism from which the nucleic acidis derived. For example, an “isolated” nucleic acid molecule, such as acDNA molecule, can be substantially free of other cellular material, orculture medium, when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention (e.g., including thesequences in Table 1), or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. For example, a nucleic acid molecule encompassing allor a portion of sequences shown in Table 1 can be isolated by thepolymerase chain reaction (PCR) using synthetic oligonucleotide primersdesigned based upon the sequences shown in Table 1.

A nucleic acid molecule of the present invention can be amplified usingcDNA, mRNA or, alternatively, genomic DNA as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to nucleic acid sequences ofthe present invention can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of the presentinvention comprises a nucleic acid molecule which is a complement of anucleic acid molecule of the present invention (e.g., including thesequences in Table 1), or a portion thereof. A nucleic acid moleculewhich is complementary to a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1), or a portionthereof, is one which is sufficiently complementary to the nucleotidesequence shown in Table 1, such that it can hybridize to the respectivenucleotide sequence shown in Table 1, thereby forming a stable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the entire length of the nucleotide sequenceshown in Table 1, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the present invention cancomprise only a portion of a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1), or a portionthereof, for example, a fragment which can be used as a probe or primeror a fragment which encodes a portion of a polypeptide of the presentinvention, e.g., those in Table 1. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1); of an anti-sensesequence of a nucleic acid molecule of the present invention (e.g.,including the sequences in Table 1); or of a mutant of a nucleic acidmolecule of the present invention (e.g., including the sequences inTable 1).

Probes based on a nucleic acid molecule of the present invention (e.g.,including the sequences in Table 1) can be used to detect transcripts orgenomic sequences encoding the same or homologous polypeptides. In oneembodiment, the probe further comprises a label group attached thereto,e.g., the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor.

A nucleic acid fragment encoding a “biologically active portion of apolypeptide of the present invention” can be prepared by isolating aportion of the nucleotide sequence of a nucleic acid molecule of thepresent invention (e.g., including the sequences in Table 1) whichencodes a polypeptide having a biological activity of a polypeptide ofthe present invention (e.g., the ability to bind to its antigenictarget, such as human Gal1), expressing the encoded portion of thepolypeptide of the present invention (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of thepolypeptide of the present invention.

In other embodiments, a nucleic acid fragment encoding a “peptideepitope of the present invention” can be prepared by isolating a portionof the nucleotide sequence of a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1) which encodes apolypeptide for which antibodies raised against the polypeptide arespecific (e.g., a human Gal1 peptide epitopes shown in Table 1).

The invention further encompasses nucleic acid molecules that differfrom nucleotide sequence(s) shown in Table 1 due to degeneracy of thegenetic code and thus encode the same polypeptides as those encoded bythe respective nucleotide sequence shown in Table 1. In anotherembodiment, an isolated nucleic acid molecule of the present inventionhas a nucleotide sequence encoding a polypeptide of the presentinvention (e.g., including the sequences in Table 1).

Nucleic acid molecules corresponding to homologues of a nucleic acidmolecule of the present invention (e.g., including the sequences inTable 1) can be isolated based on their homology to the nucleic acidsdisclosed herein using the cDNAs disclosed herein, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe present invention is at least 15, 20, 25, 30 or more nucleotides inlength and hybridizes under stringent conditions to the nucleic acidmolecule comprising a nucleic acid molecule of the present invention(e.g., including the sequences in Table 1).

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A non-limiting example of stringenthybridization conditions includes hybridization in 4× or 6× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in4×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 1×SSC, at about 65-70° C. A further non-limiting example ofstringent hybridization conditions includes hybridization at 6×SSC at45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Anon-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or hybridization in1×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 0.3×SSC, at about 65-70° C. A non-limiting example of reducedstringency hybridization conditions includes hybridization in 4× or6×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus50% formamide at about 40-45° C.) followed by one or more washes in 2×,at about 50-60° C. Ranges intermediate to the above-recited values,e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassedby the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCland 15 mM sodium citrate) in the hybridization and wash buffers; washesare performed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m) (° C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number of bases in thehybrid, and [Na⁺] is the concentration of sodium ions in thehybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional non-limitingexample of stringent hybridization conditions is hybridization in0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed by one or morewashes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Church and Gilbert(1984) Proc. Natl. Acad. Sci. USA 81:1991-1995 (or alternatively0.2×SSC, 1% SDS).

The skilled artisan will further appreciate that changes can beintroduced by mutation into a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1), thereby leading tochanges in the amino acid sequence of the encoded polypeptides of thepresent invention, without altering the functional ability of thepolypeptides. For example, nucleotide substitutions leading to aminoacid substitutions at “non-essential” amino acid residues can be made ina nucleic acid molecule of the present invention (e.g., including thesequences in Table 1). A “non-essential” amino acid residue is a residuethat can be altered from a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1) without alteringthe biological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare conserved among the polypeptides of the present invention, e.g.,those required for binding of the polypeptides to its target antigen,are predicted to be particularly unamenable to alteration.

Accordingly, another aspect of the present invention pertains to nucleicacid molecules encoding polypeptides of the present invention (e.g.,including the sequences in Table 1) that contain changes in amino acidresidues that are not essential for activity. Such polypeptides differin amino acid sequence from the sequences in Table 1, or portionsthereof, yet retain biological activity. In one embodiment, the isolatednucleic acid molecule comprises a nucleotide sequence encoding apolypeptide, wherein the polypeptide comprises an amino acid sequence atleast about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the sequences in Table 1, or portionsthereof.

An isolated nucleic acid molecule encoding a polypeptide identical tothe polypeptides of the sequences in Table 1, or portions thereof, canbe created by introducing one or more nucleotide substitutions,additions or deletions into the nucleotide sequence of the sequences inTable 1, or portions thereof, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedpolypeptide. Mutations can be introduced into nucleic acid molecules ofthe present invention (e.g., including the sequences in Table 1) bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. In one embodiment, conservative amino acid substitutionsare made at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a polypeptide of thepresent invention (e.g., including the sequences in Table 1) can bereplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a nucleic acid molecule(s) ofthe present invention (e.g., including the sequences in Table 1), suchas by saturation mutagenesis, and the resultant mutants can be screenedfor biological activity to identify mutants that retain activity.Following mutagenesis of a nucleic acid molecule of the presentinvention (e.g., including the sequences in Table 1), the encodedpolypeptide can be expressed recombinantly and the activity of thepolypeptide can be determined.

In one embodiment, a mutant polypeptide of the present invention can beassayed for the ability to bind to and/or modulate the activity of Gal1.

Yet another aspect of the present invention pertains to isolated nucleicacid molecules encoding fusion proteins. Such nucleic acid molecules,comprising at least a first nucleotide sequence encoding a polypeptideof the present invention (e.g., including the sequences in Table 1)operatively linked to a second nucleotide sequence encoding apolypeptide of the present invention (e.g., including the sequences inTable 1) can be prepared by standard recombinant DNA techniques.

The expression characteristics of a nucleic acid molecules of thepresent invention (e.g., including the sequences in Table 1) within acell line or microorganism may be modified by inserting a heterologousDNA regulatory element into the genome of a stable cell line or clonedmicroorganism such that the inserted regulatory element is operativelylinked with a nucleic acid molecule of the present invention (e.g.,including the sequences in Table 1). For example, a heterologousregulatory element may be inserted into a stable cell line or clonedmicroorganism, such that it is operatively linked with a nucleic acidmolecule of the present invention (e.g., including the sequences inTable 1), using techniques, such as targeted homologous recombination,which are well known to those of skill in the art, and described, e.g.,in Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667,published May 16, 1991.

2. Isolated Polypeptide Molecules

Another aspect of the present invention pertains to isolatedpolypeptides of the present invention (e.g., including the sequences inTable 1) and biologically active portions thereof. In one embodiment,polypeptides of the present invention (e.g., including the sequences inTable 1), and biologically active portions thereof can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment,polypeptides of the present invention (e.g., including the sequences inTable 1), and biologically active portions thereof are produced byrecombinant DNA techniques. Alternatively, polypeptides of the presentinvention (e.g., including the sequences in Table 1), and biologicallyactive portions thereof can be chemically synthesized using standardpeptide synthesis techniques.

As used herein, a “biologically active portion” of polypeptide(s) of thepresent invention (e.g., including the sequences in Table 1) includepolypeptides which participate in an interaction between Gal1 and anon-Gal1 molecule. Biologically active portions of a polypeptide(s) ofthe present invention (e.g., including the sequences in Table 1) includepeptides comprising amino acid sequences sufficiently identical to orderived from the amino acid sequence of polypeptide(s) of the presentinvention (e.g., including the sequences in Table 1), which includefewer amino acids than the respective, full length polypeptide(s) of thepresent invention (e.g., including the sequences in Table 1), andexhibit at least one activity of the respective polypeptide(s) of thepresent invention (e.g., including the sequences in Table 1). In oneembodiment, biologically active portions comprise a domain or motif withthe ability to specifically bind Gal1 according to the antigen,respectively, to which it was raised or designed to bind.

In another embodiment, polypeptide(s) of the present invention (e.g.,including the sequences in Table 1) has an amino acid sequence shown inTable 1. In other embodiments, the polypeptide is substantiallyidentical to polypeptide(s) shown in Table 1, and retains the functionalactivity of the respective polypeptide(s) shown in Table 1, yet differsin amino acid sequence due to mutagenesis, as described in detailherein. Accordingly, in another embodiment, a polypeptide(s) of thepresent invention is a polypeptide which comprises an amino acidsequence at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96% 97%, 98%, 99%, 99.5%, or 99.9% or more identical to apolypeptide(s) shown in Table 1.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Inone embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%,or 99.9% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” comprises a polypeptide(s) ofthe present invention (e.g., including the sequences in Table 1)operatively linked to a polypeptide not of the present invention. A“polypeptide(s) of the present invention” refers to a polypeptide havingan amino acid sequence corresponding to a polypeptide shown in Table 1,whereas a “polypeptide not of the present invention” refers to apolypeptide not having an amino acid sequence corresponding to apolypeptide which is not substantially homologous to a polypeptide shownin Table 1, e.g., a polypeptide which is different from a polypeptideshown in Table 1 and which is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the polypeptide(s) of the present inventionand the polypeptide(s) not of the present invention are fused in-frameto each other. The polypeptide(s) not of the present invention can befused to the N-terminus or C-terminus of the polypeptide(s) of thepresent invention and corresponds to a moiety that alters thesolubility, binding affinity, stability, or valency of thepolypeptide(s) of the present invention. In a preferred embodiment, thefusion protein comprises at least one biologically active portion of aGal1 molecule, e.g., the carbohydrate recognition domain (CRD).

A chimeric or fusion polypeptide(s) of the present invention (e.g.,including the sequences in Table 1) can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, Ausubel et al.,eds., John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide).

In one embodiment, the second peptide may optionally correspond to amoiety that alters the solubility, affinity, stability or valency of thefirst peptide, for example, an immunoglobulin constant region.Preferably, the first peptide consists of a portion of Gal1 thatcomprises at least one biologically active portion of a Gal1 molecule,e.g., the carbohydrate recognition domain (CRD). In another preferredembodiment, the first peptide consists of a portion of a biologicallyactive molecule (e.g. the extracellular portion of the polypeptide orthe ligand binding portion). The second peptide can include animmunoglobulin constant region, for example, a human Cγ1 domain or Cγ4domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, or humanIgCγ4, see e.g., Capon et al. U.S. Pat. Nos. 5,116,964; 5,580,756;5,844,095 and the like, incorporated herein by reference). Such constantregions may retain regions which mediate effector function (e.g. Fcreceptor binding) or may be altered to reduce effector function. Aresulting fusion protein may have altered solubility, binding affinity,stability and/or valency (i.e., the number of binding sites availableper polypeptide) as compared to the independently expressed firstpeptide, and may increase the efficiency of protein purification. Fusionproteins and peptides produced by recombinant techniques can be secretedand isolated from a mixture of cells and medium containing the proteinor peptide. Alternatively, the protein or peptide can be retainedcytoplasmically and the cells harvested, lysed and the protein isolated.A cell culture typically includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.Protein and peptides can be isolated from cell culture media, hostcells, or both using techniques known in the art for purifying proteinsand peptides. Techniques for transfecting host cells and purifyingproteins and peptides are known in the art.

In another embodiment, the fusion protein is a GST fusion protein with apolypeptide(s) of the present invention. Such fusion proteins canfacilitate the purification of recombinant polypeptides of the presentinvention. In another embodiment, the fusion protein contains aheterologous signal sequence at its N-terminus. In yet anotherembodiment, the fusion protein contains a cytotoxic moiety (e.g.,toxin). In certain host cells (e.g., mammalian host cells), expressionand/or secretion of polypeptide(s) of the present invention can beincreased through use of a heterologous signal sequence.

The fusion proteins of the present invention can be used as immunogensto produce antibodies in a subject. Such antibodies may be used topurify the respective natural polypeptides from which the fusionproteins were generated, or in screening assays to identify polypeptideswhich inhibit the interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof.

The amino acid sequences of polypeptide(s) of the present invention(e.g., including the sequences in Table 1) identified herein will enablethose of skill in the art to produce polypeptides corresponding topolypeptide(s) of the present invention (e.g., including the sequencesin Table 1). Such polypeptides can be produced in prokaryotic oreukaryotic host cells by expression of polynucleotides encoding apolypeptide(s) of the present invention (e.g., including the sequencesin Table 1). Alternatively, such peptides can be synthesized by chemicalmethods. Methods for expression of heterologous polypeptides inrecombinant hosts, chemical synthesis of polypeptides, and in vitrotranslation are well known in the art and are described further inManiatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed.,Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology,Volume 152, Guide to Molecular Cloning Techniques (1987), AcademicPress, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc.91:501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11:255; Kaiser etal. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent,S. B. H. (1988) Annu Rev. Biochem. 57:957; and Offord, R. E. (1980)Semisynthetic Proteins, Wiley Publishing, which are incorporated hereinby reference).

In another aspect of this invention, peptides or peptide mimetics can beused to antagonize or promote the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof. In one embodiment, variants of Gal1 whichfunction as a modulating agent for the respective full length protein,can be identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, for antagonist activity. In one embodiment, avariegated library of Gal1 variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of Gal1 variants can be produced, forinstance, by enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential polypeptide sequences is expressible as individualpolypeptides containing the set of polypeptide sequences therein. Thereare a variety of methods which can be used to produce libraries ofpolypeptide variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential polypeptide sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of a polypeptide coding sequence canbe used to generate a variegated population of polypeptide fragments forscreening and subsequent selection of variants of a given polypeptide.In one embodiment, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of a polypeptidecoding sequence with a nuclease under conditions wherein nicking occursonly about once per polypeptide, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of thepolypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of polypeptides. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofGal1 (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;Delagrave et al. (1993) Protein Eng. 6(3):327-331). In one embodiment,cell based assays can be exploited to analyze a variegated polypeptidelibrary. For example, a library of expression vectors can be transfectedinto a cell line which ordinarily synthesizes Gal1. The transfectedcells are then cultured such that the full length polypeptide and aparticular mutant polypeptide are produced and the effect of expressionof the mutant on the full length polypeptide activity in cellsupernatants can be detected, e.g., by any of a number of functionalassays. Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of full length polypeptideactivity, and the individual clones further characterized.

Systematic substitution of one or more amino acids of a polypeptideamino acid sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides comprising a polypeptide amino acidsequence of interest or a substantially identical sequence variation canbe generated by methods known in the art (Rizo and Gierasch (1992) Annu.Rev. Biochem. 61:387, incorporated herein by reference); for example, byadding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The amino acid sequences disclosed herein will enable those of skill inthe art to produce polypeptides corresponding peptide sequences andsequence variants thereof. Such polypeptides can be produced inprokaryotic or cukaryotic host cells by expression of polynucleotidesencoding the peptide sequence, frequently as part of a largerpolypeptide. Alternatively, such peptides can be synthesized by chemicalmethods. Methods for expression of heterologous proteins in recombinanthosts, chemical synthesis of polypeptides, and in vitro translation arewell known in the art and are described further in Maniatis et al.Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold SpringHarbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; ChaikenI. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989)Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H.(1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980)Semisynthetic Proteins, Wiley Publishing, which are incorporated hereinby reference).

In another aspect of the present invention, peptides are provided inwhich the peptides have an amino acid sequence identical or similar tothe Gal1 binding site of its natural binding partner(s) or a fragment(s)thereof. In one embodiment, the peptide competes with a Gal1 polypeptideor a fragment thereof for binding its natural binding partner(s) or afragment(s) thereof. In a preferred embodiment, the peptide carriescarbohydrate moieties recognized by a Gal1 polypeptide or a fragmentthereof and said peptide competes with the Gal1 polypeptide or afragment thereof for binding the Gal1 natural binding partner(s) or afragment(s) thereof.

Peptides can be produced, typically by direct chemical synthesis, andused e.g., as antagonists of the interactions between a Gal1 polypeptideor a fragment thereof and its natural binding partner(s) or afragment(s) thereof. Peptides can be produced as modified peptides, withnonpeptide moieties attached by covalent linkage to the N-terminusand/or C-terminus. In certain preferred embodiments, either thecarboxy-terminus or the amino-terminus, or both, are chemicallymodified. The most common modifications of the terminal amino andcarboxyl groups are acetylation and amidation, respectively.Amino-terminal modifications such as acylation (e.g., acetylation) oralkylation (e.g., methylation) and carboxy-terminal-modifications suchas amidation, as well as other terminal modifications, includingcyclization, can be incorporated into various embodiments of the presentinvention. Certain amino-terminal and/or carboxy-terminal modificationsand/or peptide extensions to the core sequence can provide advantageousphysical, chemical, biochemical, and pharmacological properties, suchas: enhanced stability, increased potency and/or efficacy, resistance toserum proteases, desirable pharmacokinetic properties, and others.Peptides disclosed herein can be used therapeutically to treat disease,e.g., by altering costimulation in a patient.

Peptidomimetics (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber andFreidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem.30:1229, which are incorporated herein by reference) are usuallydeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides can be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as a human Gal1 polypeptide or afragment thereof, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH2NH—,—CH2S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and—CH2SO—, by methods known in the art and further described in thefollowing references: Spatola, A. F. in “Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins” Weinstein, B., ed., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, “Peptide Backbone Modifications” (general review); Morley, J.S. (1980) Trends Pharm. Sci. pp. 463-468 (general review); Hudson, D. etal. (1979) Int. J. Pept. Prot. Res. 14:177-185 (—CH2NH—, CH2CH2-);Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249 (—CH2-S); Hann, M.M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314 (—CH—CH—, cis andtrans); Almquist, R. G. et al. (190) J. Med. Chem. 23:1392-1398(—COCH₂—); Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533(—COCH2-); Szelke, M. et al. European Appln. EP 45665 (1982) CA:97:39405 (1982) (—CH(OH)CH2-); Holladay, M. W. et al. (1983) TetrahedronLett. (1983) 24:4401-4404 (—C(OH)CH2-); and Hruby, V. J. (1982) LiftSci. (1982) 31:189-199 (—CH2-S—); each of which is incorporated hereinby reference. In one embodiment, the non-peptide linkage is —CH2NH—.Such peptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics usually involves covalent attachmentof one or more labels, directly or through a spacer (e.g., an amidegroup), to non-interfering position(s) on the peptidomimetic that arepredicted by quantitative structure-activity data and/or molecularmodeling. Such non-interfering positions generally are positions that donot form direct contacts with the macropolypeptides(s) to which thepeptidomimetic binds to produce the therapeutic effect. Derivitization(e.g., labeling) of peptidomimetics should not substantially interferewith the desired biological or pharmacological activity of thepeptidomimetic.

Similarly, glycan-related compounds and/or glycomimetics can be usedaccording to the methods of the present invention and according to wellknown methods in the art (see, e.g., U.S. Pat. Pub. 20080200406,20080112955, and 2004092015). For example, glycan-related compounds orglycomimetic analogs of proteins or peptides described herein can beused to modulate immune responses and/or hypoxia associatedangiogenesis. The terms related to any glycosidic structure,disaccharide, trisaccharide, tetrasaccharide, pentasaccharide or higherorder saccharide structure, branched or linear, substituted orunsubstituted by other chemical groups. In some embodiments, proteins,peptides, and antibodies may be glycosylated such that the glycosidicstructure are recognized by glycosidic and/or glycoylated proteinantibodies.

For example, the glycan can be a glycoaminoacid, a glycopeptide, aglycolipid, a glycoaminoglycan (GAG), a glycoprotein, a whole cell, acellular component, a glycoconjugate, a glycomimetic, aglycophospholipid anchor (GPI), glycosyl phosphatidylinositol(GPI)-linked glycoconjugates, bacterial lipopolysaccharides andendotoxins. The glycans can also include N-glycans, O-glycans,glycolipids and glycoproteins. The glycans can also include 2 or moresugar units. Any type of sugar unit can be present in the glycans of theinvention, including, for example, allose, altrose, arabinose, glucose,galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose,talose, xylose, or other sugar units. The figures provided herein listother examples of sugar units that can be used in the glycans of theinvention. Such sugar units can have a variety of modifications andsubstituents. Some examples of the types of modifications andsubstituents contemplated are provided in the figures herein. Forexample, sugar units can have a variety of substituents in place of thehydroxy (—OH), carboxylate (—COO⁻), and methylenehydroxy (—CH₂—OH)substituents. Thus, lower alkyl moieties can replace any of the hydrogenatoms from the hydroxy (—OH), carboxylic acid (—COOH) andmethylenehydroxy (—CH₂—OH) substituents of the sugar units in theglycans of the invention. For example, amino acetyl (—NH—CO—CH₃) canreplace any of the hydroxy or hydrogen atoms from the hydroxy (—OH),carboxylic acid (—COOH) and methylenehydroxy (—CH₂—OH) substituents ofthe sugar units in the glycans of the invention. N-acetylneuraminic acidcan replace any of the hydrogen atoms from the hydroxy (—OH), carboxylicacid (—COOH) and methylenehydroxy (—CH₂—OH) substituents of the sugarunits in the glycans of the invention. Sialic acid can replace any ofthe hydrogen atoms from the hydroxy (—OH), carboxylic acid (—COOH) andmethylenehydroxy (—CH₂—OH) substituents of the sugar units in theglycans of the invention. Amino or lower alkyl amino groups can replaceany of the OH groups on the hydroxy (—OH), carboxylic acid (—COOH) andmethylenehydroxy (—CH₂—OH) substituents of the sugar units in theglycans of the invention. Sulfate (—SO₄ ⁻) or phosphate (—PO₄ ⁻) canreplace any of the OH groups on the hydroxy (—OH), carboxylic acid(—COON) and methylenehydroxy (—CH₂—OH) substituents of the sugar unitsin the glycans of the invention. Hence, substituents that can be presentinstead of, or in addition to, the substituents typically present on thesugar units include N-acetyl, N-acetylneuraminic acid, oxy (O), sialicacid, sulfate (—SO₄ ⁻), phosphate (—PO₄ ⁻), lower alkoxy, loweralkanoyloxy, lower acyl, and/or lower alkanoylaminoalkyl.

The following definitions are used, unless otherwise described: Alkyl,alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups;but reference to an individual radical such as “propyl” embraces onlythe straight chain radical, when a branched chain isomer such as“isopropyl” has been specifically referred to. Halo is fluoro, chloro,bromo, or iodo.

Specifically, lower alkyl refers to (C₁-C₆)alkyl, which can be methyl,ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl,or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl,or cyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can be cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy.

It will be appreciated by those skilled in the art that the glycans ofthe invention having one or more chiral centers may exist in and beisolated in optically active and racemic forms. Some compounds mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a glycan of the invention,it being well known in the art how to prepare optically active forms(for example, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase).

3. Anti-Gal1 Antibodies

Without being bound by theory, and offered to improve the understandingof the disclosed invention, it is believed that the antibodies of thepresent invention are unique relative to known Gal1 binding antibodieswithin at least one of the CDRs (complementarity determining regions)which participate in binding to the Gal1 polypeptide. This belief isbased in part on the well known structural arrangement of elements,including the CDR containing hypervariable regions, of an antibody'sstructure. Antibodies of the present invention may also differ fromknown Gal1 binding antibodies at more than one CDR and/or at more thanone amino acid position within one or more CDR. These differences mayprovide the antibodies of the disclosed invention with thecharacteristic of binding to a different epitope than previousantibodies against Gal1 so as, for example, to be specific to human Gal1(i.e., not cross-reactive with Gal1 molecules in other species).Accordingly, the anti-human GAL1 antibodies of the present inventionrecognize human GAL1 with higher specificity and sensitivity relative toknown GAL1 antibodies. Such antibodies are suitable for, among otheruses, Western blotting (or immunoblotting), immunohistochemistry (IHC),detection of denatured or fixed forms of Gal1, ELISA assays, and RIAassays.

The antibodies of the present invention and antigen-binding fragmentsthereof may also inhibit Gal1 activity and so act as Gal1 inhibitors.Such antibodies, and fragments, may be used to both detect the presenceof Gal1 and to inhibit Gal1 activity without the need for introductionof an additional Gal1 inhibitor. Alternatively, a Gal1 inhibitoryantibody or antigen-binding fragment thereof may be used in combinationwith another Gal1 inhibitor, such as in a composition for inhibitingGal1 activity or as administered, separately or in combination, to asubject as part of a method to inhibit Gal1 activity.

Monoclonal antibodies of the present invention can be produced using avariety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibodies also can be employed, e.g., viral or oncogenic transformationof B lymphocytes, phage display technique using libraries of humanantibody genes.

One method for generating hybridomas which produce monoclonal antibodiesof the present invention is the murine system. Hybridoma production inthe mouse is well known in the art, including immunization protocols andtechniques for isolating and fusing immunized splenocytes.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide immunogen. An antigenic peptide ofGal1 comprises at least 8 amino acid residues and encompasses an epitopepresent in the respective full length molecule such that an antibodyraised against the peptide forms a specific immune complex with therespective full length molecule. Preferably, the antigenic peptidecomprises at least 10 amino acid residues. Preferred epitopesencompassed by the antigenic peptides are regions of Gal1 that mediateligand specific carbohydrate binding, e.g., the Gal1 carbohydraterecognition domain, amino acids 30 to 90 of human Gal1, and amino acids62 to 86 of human Gal1. In one embodiment such epitopes can be specificfor a given polypeptide molecule from one species, such as mouse orhuman (i.e., an antigenic peptide that spans a region of the polypeptidemolecule that is not conserved across species is used as immunogen; suchnon conserved residues can be determined using an alignment such as thatprovided herein). The polypeptide antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody directed against the antigen canbe isolated from the mammal (e.g., from the blood) and further purifiedby well known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. 76:2927-31; andYeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human Bcell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally Kenneth, R. H. in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al.(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with an immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds to thepolypeptide antigen, preferably specifically.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-Gal1 monoclonal antibody (see, e.g., Galfre, G. et al. (1977)Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra;Kenneth (1980) supra). Moreover, the ordinary skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof mycloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from theAmerican Type Culture Collection (ATCC), Rockville, Md. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the present invention are detected byscreening the hybridoma culture supernatants for antibodies that bind agiven polypeptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for one of the above described polypeptides can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe appropriate polypeptide to thereby isolate immunoglobulin librarymembers that bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening an antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY)9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson etal. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci.USA 89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al.(1990) Nature 348:552-554.

Additionally, recombinant anti-Gal1 antibodies, such as chimeric,composite, and humanized monoclonal antibodies, which can be made usingstandard recombinant DNA techniques, can be generated. Such chimeric,composite, and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in Robinson et al. International Patent PublicationPCT/US86/02269; Akira et al. European Patent Application 184,187;Taniguchi, M. European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) CancerRes. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable generic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

In another embodiment, human monoclonal antibodies directed against Gal1can be generated using transgenic or transchromosomal mice carryingparts of the human immune system rather than the mouse system. In oneembodiment, transgenic mice, referred to herein as “HuMAb mice” whichcontain a human immunoglobulin gene miniloci that encodes unrearrangedhuman heavy (μ and γ) and κ light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859).Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994),supra; reviewed in Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65 93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci. 764:536 546). The preparation of HuMAb mice is describedin Taylor, L. et al. (1992) Nucleic Acids Research 20:6287 6295; Chen,J. et al. (1993) International Immunology 5: 647 656; Tuaillon et al.(1993) Proc. Natl. Acad. Sci. USA 90:3720 3724; Choi et al. (1993)Nature Genetics 4:117 123; Chen, J. et al. (1993) EMBO J. 12: 821 830;Tuaillon et al. (1994) J. Immunol. 152:2912 2920; Lonberg et al., (1994)Nature 368(6474): 856 859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49 101; Taylor, L. et al. (1994) InternationalImmunology 6: 579 591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65 93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci. 764:536 546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845 851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharmInternational; U.S. Pat. No. 5,545,807 to Surani et al.; InternationalPublication Nos. WO 98/24884, published on Jun. 11, 1998; WO 94/25585,published Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO92/22645, published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992.

In another embodiment, an antibody for use in the invention is abispecific antibody. A bispecific antibody has binding sites for twodifferent antigens within a single antibody polypeptide. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies. Examplesof bispecific antibodies produced by a hybrid hybridoma or a trioma aredisclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have beenconstructed by chemical means (Staerz et al. (1985) Nature 314:628, andPerez et al. (1985) Nature 316:354) and hybridoma technology (Staerz andBevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan(1986) Immunol. Today 7:241). Bispecific antibodies are also describedin U.S. Pat. No. 5,959,084. Fragments of bispecific antibodies aredescribed in U.S. Pat. No. 5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. The antibody component can bind to Gal1. In one embodiment,the bispecific antibody could specifically bind to both Gal1 and anon-Gal1 molecule.

Yet another aspect of the present invention pertains to anti-Gal1polypeptide antibodies that are obtainable by a process comprising,immunizing an animal with an immunogenic Gal1 polypeptide or animmunogenic portion thereof (e.g., Gal1 polypeptides shown in Table 1),and then isolating from the animal antibodies that specifically bind tothe polypeptide.

In still another aspect of the present invention, partial or knownantibody sequences can be used to generate and/or express newantibodies. Antibodies interact with target antigens predominantlythrough amino acid residues that are located in the six heavy and lightchain complementarity determining regions (CDRs). For this reason, theamino acid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323 327; Jones, P. et al., 1986, Nature 321:522 525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029 10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline or non-germline antibody gene sequences. These germlinesequences will differ from mature antibody gene sequences because theywill not include completely assembled variable genes, which are formedby V(D)J joining during B cell maturation. Germline gene sequences willalso differ from the sequences of a high affinity secondary repertoireantibody at individual evenly across the variable region. For example,somatic mutations are relatively infrequent in the amino-terminalportion of framework region. For example, somatic mutations arerelatively infrequent in the amino terminal portion of framework region1 and in the carboxy-terminal portion of framework region 4.Furthermore, many somatic mutations do not significantly alter thebinding properties of the antibody. For this reason, it is not necessaryto obtain the entire DNA sequence of a particular antibody in order torecreate an intact recombinant antibody having binding propertiessimilar to those of the original antibody. Partial heavy and light chainsequence spanning the CDR regions is typically sufficient for thispurpose. The partial sequence is used to determine which germline and/ornon-germline variable and joining gene segments contributed to therecombined antibody variable genes. The germline and/or non-germlinesequence is then used to fill in missing portions of the variableregions. Heavy and light chain leader sequences are cleaved duringprotein maturation and do not contribute to the properties of the finalantibody. To add missing sequences, cloned cDNA sequences can becombined with synthetic oligonucleotides by ligation or PCRamplification. Alternatively, the entire variable region can besynthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons. The process can also be used to screen libraries of particularimmunoglobulin encoding sequences in one species (e.g., human) to designcognate immunoglobulin encoding sequences from known antibody sequencein another species (e.g., mouse).

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266L19867019870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region in the PCR amplification to generate fragments that caneasily be cloned into the expression vector constructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains. Plasmids for this use are known in theart. Fully human and chimeric antibodies of the present invention alsoinclude IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmidscan be constructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

Thus, in another aspect of the present invention, the structuralfeatures of known, non-human or human antibodies (e.g., a mouseanti-human Gal1 antibody) can be used to create structurally relatedhuman anti-human Gal1 antibodies that retain at least one functionalproperty of the antibodies of the present invention, such as binding toGal1. Another functional property includes inhibiting binding of theoriginal known, non-human or human antibodies in a competition ELISAassay. In addition, one or more CDR or variable regions of the presentinvention (e.g., including the sequences of Table 1, or portionsthereof) can be combined recombinantly with known human frameworkregions and CDRs to create additional, recombinantly-engineered, humananti-Gal1 antibodies of the present invention.

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the present invention prepared as set forth abovepreferably comprise the heavy and light chain CDR3s of variable regionsof the present invention (e.g., including the sequences of Table 1, orportions thereof). The antibodies further can comprise the CDR2s ofvariable regions of the present invention (e.g., including the sequencesof Table 1, or portions thereof). The antibodies further can comprisethe CDR1s of variable regions of the present invention (e.g., includingthe sequences of Table 1, or portions thereof). In other embodiments,the antibodies can comprise any combinations of the CDRs.

The CDR1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those of variableregions of the present invention (e.g., including the sequences of Table1, or portions thereof) disclosed herein. However, the ordinarilyskilled artisan will appreciate that some deviation from the exact CDRsequences may be possible while still retaining the ability of theantibody to bind Gal1 effectively (e.g., conservative sequencemodifications). Accordingly, in another embodiment, the engineeredantibody may be composed of one or more CDRs that are, for example, 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical to one or more CDRs of the present invention (e.g.,including the sequences of Table 1, or portions thereof).

In another aspect, the present invention features anti-Gal1 antibodiesconjugated to a therapeutic moiety, such as a cytotoxin, a drug, and/ora radioisotope. When conjugated to a cytotoxin, these antibodyconjugates are referred to as “immunotoxins.” A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells.Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating a relateddisorder, such as a cancer.

Conjugated anti-Gal1 antibodies can be used diagnostically orprognostically to monitor polypeptide levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,P-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

The antibody conjugates of the present invention can be used to modify agiven biological response. The therapeutic moiety is not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-.gamma.; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophagecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other cytokines or growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243 56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623 53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303 16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119 58 (1982).

4. Recombinant Expression Vectors and Host Cells

Another aspect of the present invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid molecule encoding apolypeptide of the present invention (e.g., including the sequences ofTable 1, or portions thereof). As used herein, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the present invention comprise anucleic acid of the present invention in a form suitable for expressionof the nucleic acid in a host cell, which means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which isoperatively linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel (1990)Methods Enzymol. 185:3-7. Regulatory sequences include those whichdirect constitutive expression of a nucleotide sequence in many types ofhost cells and those which direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the present invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein.

The recombinant expression vectors of the present invention can bedesigned for expression of polypeptides of the present invention (e.g.,including the sequences of Table 1, or portions thereof) in prokaryoticor eukaryotic cells. For example, the polypeptides can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors), yeast cells, or mammalian cells. Suitable hostcells are discussed further in Goeddel (1990) supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a polypeptide encoded therein,usually to the amino terminus of the recombinant polypeptide. Suchfusion vectors typically serve three purposes: 1) to increase expressionof recombinant polypeptide; 2) to increase the solubility of therecombinant polypeptide; and 3) to aid in the purification of therecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 1 Id (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression in E. coliis to express the polypeptide in host bacteria with impaired capacity toproteolytically cleave the recombinant polypeptide (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of thepresent invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, polypeptides of the present invention (e.g., includingthe sequences of Table 1, or portions thereof) can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of polypeptides in cultured insect cells (e.g.,Sf 9 cells) include the pAc 'series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the present invention(e.g., including the sequences of Table 1, or portions thereof) isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the .alpha.-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

Another aspect of the present invention pertains to host cells intowhich a nucleic acid molecule of the present invention (e.g., Table 1)is introduced within a recombinant expression vector or a nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present invention (e.g., including the sequences ofTable 1, or portions thereof) can be expressed in bacterial cells suchas E. coli, insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a Gal1 polypeptide or anti-Gal1 antibodypolypeptide or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the present invention, such as a prokaryotic oreukaryotic host cell in culture, can be used to produce (i.e., express)a polypeptide of the present invention (e.g., including the sequences ofTable 1, or portions thereof). Accordingly, the invention furtherprovides methods for producing a polypeptide of the present invention(e.g., including the sequences of Table 1, or portions thereof) usingthe host cells of the present invention. In one embodiment, the methodcomprises culturing the host cell of the present invention (into which arecombinant expression vector encoding a polypeptide of the presentinvention (e.g., including the sequences of Table 1, or portionsthereof) has been introduced) in a suitable medium such that apolypeptide of the present invention (e.g., including the sequences ofTable 1, or portions thereof) is produced. In another embodiment, themethod further comprises isolating a polypeptide of the presentinvention (e.g., including the sequences of Table 1, or portionsthereof) from the medium or the host cell.

The host cells of the present invention can also be used to producenon-human transgenic animals, as described below.

5. Other Agents

Also encompassed by the present invention are small molecules which canmodulate (either enhance or inhibit) interactions, e.g., theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. The small moleculesof the present invention can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Compounds can be screened in cell based or non-cell basedassays. Compounds can be screened in pools (e.g. multiple compounds ineach testing sample) or as individual compounds. In one embodiment, thesmall molecule binds to the binding site involved in interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof.

Also provided herein are compositions comprising one or more nucleicacids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20or more small nucleic acids or antisense oligonucleotides or derivativesthereof, wherein said small nucleic acids or antisense oligonucleotidesor derivatives thereof in a cell specifically hybridize (e.g., bind)under cellular conditions, with cellular nucleic acids (e.g., Gal1 mRNAor a fragment thereof). In one embodiment, expression of the smallnucleic acids or antisense oligonucleotides or derivatives thereof in acell can enhance or upregulate one or more biological activitiesassociated with the corresponding wild-type, naturally occurring, orsynthetic small nucleic acids. In another embodiment, expression of thesmall nucleic acids or antisense oligonucleotides or derivatives thereofin a cell can inhibit expression or biological activity of cellularnucleic acids and/or proteins, e.g., by inhibiting transcription,translation and/or small nucleic acid processing of, for example, theGal1 gene or gene products or fragment(s) thereof. In one embodiment,the small nucleic acids or antisense oligonucleotides or derivativesthereof are small RNAs (e.g., microRNAs) or complements of small RNAs.In another embodiment, the small nucleic acids or antisenseoligonucleotides or derivatives thereof can be single or double strandedand are at least six nucleotides in length and are less than about 1000,900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, or 10 nucleotides in length. In anotherembodiment, a composition may comprise a library of nucleic acidscomprising or capable of expressing small nucleic acids or antisenseoligonucleotides or derivatives thereof, or pools of said small nucleicacids or antisense oligonucleotides or derivatives thereof A pool ofnucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleicacids comprising or capable of expressing small nucleic acids orantisense oligonucleotides or derivatives thereof.

In one embodiment, binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” refers to the range of techniquesgenerally employed in the art, and includes any process that relies onspecific binding to oligonucleotide sequences.

Small nucleic acid and/or antisense constructs of the methods andcompositions presented herein can be delivered, for example, as anexpression plasmid which, when transcribed in the cell, produces RNAwhich is complementary to at least a unique portion of cellular nucleicacids (e.g., small RNAs, mRNA, and/or genomic DNA). Alternatively, smallnucleic acids and/or antisense constructs are oligonucleotide probesthat are generated ex vivo and which, when introduced into the cell,results in hybridization with cellular nucleic acids (e.g., Gal1 mRNA ora fragment thereof). Such oligonucleotide probes are preferably modifiedoligonucleotides that are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as small nucleic acids and/orantisense oligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

Antisense approaches may involve the design of oligonucleotides (eitherDNA or RNA) that are complementary to cellular nucleic acids (e.g., Gal1mRNA or a fragment thereof). Absolute complementarity is not required.In the case of double-stranded antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches with anucleic acid (e.g., RNA) it may contain and still form a stable duplex(or triplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofgenes could be used in an antisense approach to inhibit translation ofendogenous mRNAs. Oligonucleotides complementary to the 5′ untranslatedregion of the mRNA may include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could also be used in accordancewith the methods and compositions presented herein. Whether designed tohybridize to the 5′, 3′ or coding region of cellular mRNAs, smallnucleic acids and/or antisense nucleic acids should be at least sixnucleotides in length, and can be less than about 1000, 900, 800, 700,600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, or 10 nucleotides in length.

Regardless of the choice of target sequence, in vitro studies may beperformed to quantitate the ability of the antisense oligonucleotide toinhibit gene expression. In one embodiment these studies utilizecontrols that distinguish between antisense gene inhibition andnonspecific biological effects of oligonucleotides. In anotherembodiment these studies compare levels of the target nucleic acid orprotein with that of an internal control nucleic acid or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

Small nucleic acids and/or antisense oligonucleotides can be DNA or RNAor chimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. Small nucleic acids and/or antisenseoligonucleotides can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,hybridization, etc., and may include other appended groups such aspeptides (e.g., for targeting host cell receptors), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.(1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988)BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon(1988), Pharm. Res. 5:539-549). To this end, small nucleic acids and/orantisense oligonucleotides may be conjugated to another molecule, e.g.,a peptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Small nucleic acids and/or antisense oligonucleotides may comprise atleast one modified base moiety which is selected from the groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxytiethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Small nucleic acids and/or antisenseoligonucleotides may also comprise at least one modified sugar moietyselected from the group including but not limited to arabinose,2-fluoroarabinose, xylulose, and hexose.

Small nucleic acids and/or antisense oligonucleotides can also contain aneutral peptide-like backbone. Such molecules are termed peptide nucleicacid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993)Nature 365:566. One advantage of PNA oligomers is their capability tobind to complementary DNA essentially independently from the ionicstrength of the medium due to the neutral backbone of the DNA. In yetanother embodiment, small nucleic acids and/or antisenseoligonucleotides comprises at least one modified phosphate backboneselected from the group consisting of a phosphorothioatc, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In a further embodiment, small nucleic acids and/or antisenseoligonucleotides are α-anomeric oligonucleotides. An α-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual b-units, the strandsrun parallel to each other (Gautier et al. (1987) Nucl. Acids Res.15:6625-6641). The oligonucleotide is a 2′-0-methylribonucleotide (Inoueet al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Small nucleic acids and/or antisense oligonucleotides of the methods andcompositions presented herein may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988) Nucl. Acids Res. 16:3209,methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Small nucleic acids and/or antisense oligonucleotides can be deliveredto cells in vivo. A number of methods have been developed for deliveringsmall nucleic acids and/or antisense oligonucleotides DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

In one embodiment, small nucleic acids and/or antisense oligonucleotidesmay comprise or be generated from double stranded small interfering RNAs(siRNAs), in which sequences fully complementary to cellular nucleicacids (e.g. mRNAs) sequences mediate degradation or in which sequencesincompletely complementary to cellular nucleic acids (e.g., mRNAs)mediate translational repression when expressed within cells. In anotherembodiment, double stranded siRNAs can be processed into single strandedantisense RNAs that bind single stranded cellular RNAs (e.g., microRNAs)and inhibit their expression. RNA interference (RNAi) is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by double-stranded RNA (dsRNA) that is homologous insequence to the silenced gene. In vivo, long dsRNA is cleaved byribonuclease III to generate 21- and 22-nucleotide siRNAs. It has beenshown that 21-nucleotide siRNA duplexes specifically suppress expressionof endogenous and heterologous genes in different mammalian cell lines,including human embryonic kidney (293) and HeLa cells (Elbashir et al.(2001) Nature 411:494-498). Accordingly, translation of a gene in a cellcan be inhibited by contacting the cell with short double stranded RNAshaving a length of about 15 to 30 nucleotides or of about 18 to 21nucleotides or of about 19 to 21 nucleotides. Alternatively, a vectorencoding for such siRNAs or short hairpin RNAs (shRNAs) that aremetabolized into siRNAs can be introduced into a target cell (see, e.g.,McManus et al. (2002) RNA 8:842; Xia et al. (2002) Nature Biotechnology20:1006; and Brummelkamp et al. (2002) Science 296:550). Vectors thatcan be used are commercially available, e.g., from OligoEngine under thename PSUPER RNAI SYSTEM™. An exemplary Gal1 shRNA target sequence isGCTGCCAGATGGATACGAA (SEQ ID NO: 5).

Ribozyme molecules designed to catalytically cleave cellular mRNAtranscripts can also be used to prevent translation of cellular mRNAs(e.g., Gal1 mRNA or a fragment thereof) and expression of cellularpolypeptides, or both (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al. (1990) Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroycellular mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach (1988) Nature 334:585-591. The ribozyme may be engineered sothat the cleavage recognition site is located near the 5′ end ofcellular mRNAs; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the methods and compositions presented herein alsoinclude RNA endoribonucleases (hereinafter “Cech-type ribozymes”) suchas the one which occurs naturally in Tetrahymena thermophile (known asthe IVS, or L-19 IVS RNA) and which has been extensively described byThomas Cech and collaborators (Zaug, et al. (1984) Science 224:574-578;Zaug, et al. (1986) Science 231:470-475; Zaug, et al. (1986) Nature324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been, et al. (1986) Cell 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The methods and compositions presented herein encompasses thoseCech-type ribozymes which target eight base-pair active site sequencesthat are present in cellular genes.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express Gal1 genes or a fragmentthereof in vivo. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenouscellular messages and inhibit translation. Because ribozymes unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription of cellular genes (e.g., the Gal1 gene or afragment thereof) are preferably single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purincs orpyrimidincs to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Small nucleic acids, antisense oligonucleotides, ribozymes, and triplehelix molecules of the methods and compositions presented herein may beprepared by any method known in the art for the synthesis of DNA and RNAmolecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides and oligoribonucleotides well known in the artsuch as for example solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors whichincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone. One of skill inthe art will readily understand that regulatable proteins, inhibitorymutants, small nucleic acids, and antisense oligonucleotides can befurther linked to another peptide or polypeptide (e.g., a heterologouspeptide), e.g., that serves as a means of protein detection.Non-limiting examples of label peptide or polypeptide moieties usefulfor detection in the invention include, without limitation, suitableenzymes such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG,MYC, HA, or HIS tags; fluorophores such as green fluorescent protein;dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers; as wellas others known in the art, for example, in Principles of FluorescenceSpectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition(July 1999).

The modulatory agents described herein (e.g. antibodies, smallmolecules, peptides, fusion proteins, or small nucleic acids) can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The compositions may contain a single such molecule oragent or any combination of modulatory agents described herein.

IV. Methods of Selecting Agents that Modulate Immune Cell Activationand/or Hypoxia Associated Angiogenesis

Another aspect of the present invention relates to methods of selectingagents (e.g., antibodies, fusion proteins, peptides, small molecules, orsmall nucleic acids) which modulate an immune response by modulating theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. Such methodsutilize screening assays, including cell based and non-cell basedassays.

In one embodiment, the invention relates to assays for screeningcandidate or test compounds which bind to, or modulate the activity of,a Gal1 polypeptide or a fragment thereof, e.g., modulate the ability ofa Gal1 polypeptide or a fragment thereof to interact with, e.g., bindto, its natural binding partner(s) or a fragment(s) thereof. In oneembodiment, a method for identifying an agent to modulate an immuneresponse and/or hypoxia associated angiogenesis entails determining theability of the agent to modulate, e.g., enhance or inhibit, theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. Such agentsinclude, without limitation, antibodies, proteins, fusion proteins andsmall molecules.

In one embodiment, a method for identifying an agent which enhances animmune response entails determining the ability of the candidate agentto inhibit the interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof. Inanother embodiment, a method for identifying an agent to decrease animmune response entails determining the ability of a candidate agent toenhance the interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof. Instill another embodiment, a method for identifying an agent whichdecreases hypoxia associated angiogenesis entails determining theability of the candidate agent of inhibit the interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof.

In one embodiment, an assay is a cell-based assay, comprising contactinga cell expressing a Gal1 polypeptide or a fragment thereof, with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the binding between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof. Determining the ability of a Gal1 polypeptide or a fragmentthereof to bind to, or interact with, a binding partner or a fragmentthereof, can be accomplished, e.g., by measuring direct binding or bymeasuring a parameter of immune cell activation and/or hypoxiaassociated angiogenesis.

For example, in a direct binding assay, a Gal1 polypeptide, a Gal1binding partner(s), or a fragment(s) thereof, can be coupled with aradioisotope or enzymatic label such that binding of the Gal1polypeptide or a fragment thereof to its natural binding partner(s) or afragment(s) thereof can be determined by detecting the labeled moleculein a complex. For example, a Gal1 polypeptide, a Gal1 bindingpartner(s), or a fragment(s) thereof, can be labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, a Gal1 polypeptide, a Gal1 binding partner(s), or afragment(s) thereof, can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interactions between a Gal1 polypeptide ora fragment thereof and its natural binding partner(s) or a fragment(s)thereof, without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof without the labeling of either a Gal1 polypeptideor a fragment thereof and its natural binding partner(s) or afragment(s) thereof (McConnell, H. M. et al. (1992) Science257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between compound and receptor.

In a preferred embodiment, determining the ability of the blockingagents (e.g. antibodies, fusion proteins, peptides, or small molecules)to antagonize the interaction between a given set of polypeptides can beaccomplished by determining the activity of one or more members of theset of interacting molecules. For example, the activity of Gal1 can bedetermined by detecting induction of a cellular second messenger (e.g.,H-Ras), detecting catalytic/enzymatic activity of an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a cellular response regulated by a Gal1polypeptide or a fragment thereof. Determining the ability of theblocking agent to bind to or interact with said polypeptide can beaccomplished by measuring the ability of an agent to modulate immuneresponses, for example, by detecting changes in type and amount ofcytokine secretion, changes in apoptosis or proliferation, changes ingene expression or activity associated with cellular identity, or byinterfering with the ability of said polypeptide to bind to antibodiesthat recognize a portion thereof.

Agents that block or inhibit interactions between a Gal1 polypeptide ora fragment thereof and its natural binding partner(s) or a fragment(s)thereof (e.g., blocking antibodies to a Gal1 polypeptide or a fragmentthereof) can be identified by their ability to inhibit immune cellproliferation, and/or effector function, induce apoptosis, or to induceanergy when added to an in vitro assay. For example, cells can becultured in the presence of an agent that stimulates signal transductionvia an activating receptor. A number of recognized readouts of cellactivation can be employed to measure, cell proliferation, apoptosis, oreffector function (e.g., antibody production, cytokine production,phagocytosis) in the presence of the activating agent. The ability of atest agent to block this activation can be readily determined bymeasuring the ability of the agent to effect a decrease inproliferation, increase apoptosis, or effector function being measured,using techniques known in the art.

A number of art-recognized methods are further known to determinewhether a candidate agent can reduce hypoxia associated angiogenesis.For example, endothelial cell adhesion and migration are known toregulate endothelial cell survival, proliferation, and motility duringnew blood vessel growth in normal and pathologic conditions that involveangiogenesis. The term “endothelial cell adhesion” as used herein refersto the adhesion of an endothelial cell to one or more components of theextracellular matrix (e.g., fibronectin, collagens I-XVIII, laminin,vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrands'sfactor, etc.), to a ligand which is expressed on the cell surface (e.g.,VCAM, ICAM, LI-CAM, VE-cadherin, integrin a2, integrin a3, etc.) and/orto another cell (e.g., another endothelial cell, to a fibroblast cell,stromal cell, tumor cell, etc.) The terms “inhibiting endothelial celladhesion” and “reducing endothelial cell adhesion” refer to reducing thelevel of adhesion of an endothelial cell to one or more components ofthe extracellular matrix (e.g., fibronectin, collagens I-XVIII, laminin,vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrands'sfactor, etc.), and/or to another cell (e.g., another endothelial cell,fibroblast cell, stromal cell, tumor cell, etc.) to a quantity which ispreferably 10% less than, more preferably 50% less than, yet morepreferably 75% than, even more preferably 90% less than, the quantity ina corresponding control endothelial cell, and most preferably is at thesame level which is observed in a control endothelial cell. A reducedlevel of endothelial cell adhesion need not, although it may, mean anabsolute absence of cell adhesion. The invention does not require, andis not limited to, methods that wholly eliminate cell adhesion. Thelevel of endothelial cells adhesion may be determined using methods wellknown in the art. The term “endothelial cell migration” as used hereinrefers to the translocation of an endothelial cell across one or morecomponents of the extracellular matrix (e.g., fibronectin, collagensI-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin,von Willebrands's factor, etc.), or along the surface of another cell(e.g., another endothelial cell, fibroblast cell, stromal cell, tumorcell, etc.).

The terms “inhibiting endothelial cell migration” and “reducingendothelial cell migration” refer to reducing the level of migration ofan endothelial cell to a quantity which is preferably 10% less than,more preferably 50% less than, yet more preferably 75% less than, andeven more preferably 90% less than, the quantity in a correspondingcontrol endothelial cells, and most preferably is at the same levelwhich is observed in a control endothelial cell. A reduced level ofendothelial cell migration need not, although it may, mean an absoluteabsence of cell migration. The invention does not require, and is notlimited to, methods that wholly eliminate cell migration. The level ofendothelial cells migration may be determined using methods well knownin the art, such as time lapse video microscopy, scratch type woundassay.

For hypoxia associated angiogenesis involving ischemia, severalart-recognized models for studying ischemia are known. These include,but are not limited to, experimentally induced rat hindlimb ischemia(see, e.g., Takeshita, S. et al., Circulation (1998) 98: 1261-63; andTakeshita, S. et al. (1994) Circulation 90(#5; part II):228-234), apartially ischemic hindlimb rabbit model (see, e.g., Hopkins, S. et al.,J. Vasc. Surg. (1998) 27: 886-894), and a chronic porcine myocardialischemia model (see, e.g., Harada, K. et al., Am. J. Physiol. (1996)270: 886-94; and Hariawala, M. et al., 1996, J. Surg. Res. 63: 77-82).Another assay includes a rabbit model of hindlimb ischemia (see, e.g.,Takeshita, S. et al., 1994, Circulation 90(#5; part II):228-234).

In yet another embodiment, an assay of the present invention is acell-free assay in which a Gal1 polypeptide or a fragment thereof, e.g.a biologically active fragment thereof, is contacted with a testcompound, and the ability of the test compound to bind to thepolypeptide, or biologically active portion thereof, is determined.Binding of the test compound to a Gal1 polypeptide or a fragmentthereof, can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the Gal1polypeptide or fragment thereof, with a Gal1 natural binding partner(s)or fragment(s) thereof, to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with the polypeptide in the assay mixture, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the test compound topreferentially bind to the polypeptide or fragment thereof, as comparedto the binding partner.

For example, a Gal1 polypeptide or a fragment thereof and its naturalbinding partner(s) or a fragment(s) thereof can be used to form an assaymixture and the ability of a polypeptide to block this interaction canbe tested by determining the ability of a Gal1 polypeptide or a fragmentthereof to bind to the Gal1 natural binding partner(s) or a fragment(s)thereof, by one of the methods described above for determining directbinding. Determining the ability of a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof canalso be accomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA) (Sjolander, S, and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705). As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological polypeptides. A Gal1 polypeptideor a fragment thereof can be immobilized on a BIAcore chip and multipleagents, e.g., blocking antibodies, fusion proteins, peptides, or smallmolecules, can be tested for binding to the immobilized Gal1 polypeptideor fragment thereof. An example of using the BIA technology is describedby Fitz et al. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., Gal1polypeptides, Gal1 binding partner(s) polypeptides, and fragmentsthereof). In the case of cell-free assays in which a membrane-bound formprotein is used (e.g., a cell surface Gal1 polypeptide or a fragmentthereof or Gal1 natural binding partner(s) or a fragment(s) thereof) itmay be desirable to utilize a solubilizing agent such that themembrane-bound form of the protein is maintained in solution. Examplesof such solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON X-100(4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol), TRITON X-114(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, THESIT®(hydroxypolyethoxydodecane), Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In one or more embodiments of the above described assay methods, it maybe desirable to immobilize either the Gal1 polypeptide, the Gal1 naturalbinding partner(s) polypeptide, or fragments thereof, to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test compound to a Gal1 polypeptide, a Gal1 natural binding partner(s)polypeptide, or fragments thereof, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/Gal1 or glutathione-S-transferase/Gal1 naturalbinding partner(s) fusion proteins, can be adsorbed onto glutathioneSEPHAROSE® (cross-linked agarose) beads (Sigma Chemical, St. Louis, Mo.)or glutathione derivatized microtiter plates, which are then combinedwith the test compound, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of Gal1 binding or activitydetermined using standard techniques.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a Gal1 or Gal1 natural bindingpartner(s) can be accomplished by determining the ability of the testcompound to modulate the expression or activity of a gene, e.g., nucleicacid, or gene product, e.g., polypeptide, that functions downstream ofGal1 or a Gal1 natural binding partner(s), e.g., a polypeptide thatfunctions downstream of the Gal1 natural binding partner(s). Forexample, levels of second messengers can be determined, the activity ofthe interactor polypeptide on an appropriate target can be determined,or the binding of the interactor to an appropriate target can bedetermined as previously described.

In another embodiment, modulators of Gal1 expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of Gal1 mRNA or polypeptide or fragments thereof in the cellis determined. The level of expression of Gal1 mRNA or polypeptide orfragments thereof in the presence of the candidate compound is comparedto the level of expression of Gal1 mRNA or polypeptide or fragmentsthereof in the absence of the candidate compound. The candidate compoundcan then be identified as a modulator of Gal1 expression based on thiscomparison. For example, when expression of Gal1 mRNA or polypeptide orfragments thereof is greater (statistically significantly greater) inthe presence of the candidate compound than in its absence, thecandidate compound is identified as a stimulator of Gal1 expression.Alternatively, when expression of Gal1 mRNA or polypeptide or fragmentsthereof is reduced (statistically significantly less) in the presence ofthe candidate compound rather than in its absence, the candidatecompound is identified as an inhibitor of Gal1 expression. Theexpression level of Gal1 mRNA or polypeptide or fragments thereof in thecells can be determined by methods described herein for detecting Gal1mRNA or polypeptide or fragments thereof.

In yet another aspect of the present invention, Gal1 polypeptides orfragments thereof can be used as “bait proteins” in a two-hybrid assayor three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother polypeptides which bind to or interact with Gal1 or fragmentsthereof (“Gal1-binding proteins”, “Gal1 binding partners”, or “Gal1-bp”)and are involved in Gal1 activity. Such Gal1-binding proteins are alsolikely to be involved in the propagation of signals by the Gal1polypeptides or Gal1 natural binding partner(s) as, for example,downstream elements of a Gal1-mediated signaling pathway. Alternatively,such Gal1-binding polypeptides may be Gal1 inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a Gal1 polypeptideis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedpolypeptide (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” polypeptides are able to interact, in vivo, forming aGal1-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes thepolypeptide which interacts with the Gal1 polypeptide.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of a Gal1 polypeptide or afragment thereof can be confirmed in vivo, e.g., in an animal such as ananimal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

V. Pharmaceutical Compositions

Gal1 modulating agents (e.g., agents that inhibit or promote theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment thereof, including, e.g.,blocking antibodies, peptides, fusion proteins, or small molecules) canbe incorporated into pharmaceutical compositions suitable foradministration to a subject. Such compositions typically comprise theantibody, peptide, fusion protein or small molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the present invention is formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerin, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (macrogolglycerol ricinoleate) (BASF, Parsippany, N.J.) or phosphatebuffered saline (PBS). In all cases, the composition should be sterileand should be fluid to the extent that easy syringeability exists. Itmust be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., blocking antibodies, peptides, fusion proteins, or smallmolecules that inhibit the interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, modulatory agents are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the present invention are dictated by, anddirectly dependent on, the unique characteristics of the activecompound, the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the present invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity of Gal1 nucleic acid, polypeptide, or fragments thereof. Anagent may, for example, be a small molecule. For example, such smallmolecules include, but are not limited to, peptides, peptidomimetics,amino acids, amino acid analogs, polynucleotides, polynucleotideanalogs, nucleotides, nucleotide analogs, organic or inorganic compounds(i.e., including heterorganic and organometallic compounds) having amolecular weight less than about 10,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 5,000grams per mole, organic or inorganic compounds having a molecular weightless than about 1,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 500 grams per mole, and salts,esters, and other pharmaceutically acceptable forms of such compounds.It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the scope of knowledge of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the present invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of the presentinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the present invention can be used for modifying agiven biological response, the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such polypeptides may include, for example, a toxin such asabrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a proteinsuch as tumor necrosis factor, alpha-interferon, beta-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al. “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The above described modulating agents may be administered it the form ofexpressible nucleic acids which encode said agents. Such nucleic acidsand compositions in which they are contained, are also encompassed bythe present invention. For instance, the nucleic acid molecules of thepresent invention can be inserted into vectors and used as gene therapyvectors. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The Gal1 molecules, e.g., the Gal1 nucleic acid molecules, polypeptides,polypeptide homologues, antibodies, and fragments thereof, describedherein can be used in one or more of the following methods: a) screeningassays; b) predictive medicine (e.g., diagnostic assays, prognosticassays, and monitoring clinical trials); and c) methods of treatment(e.g., therapeutic and prophylactic, e.g., by up- or down-modulating theimmune response and/or downregulating hypoxia associated angiogenesis).As described herein, a Gal1 polypeptide or fragment thereof of thepresent invention has one or more of the following activities: 1) bindsto and/or modulates the activity of its natural binding partner(s), 2)modulates intra- or intercellular signaling, 3) modulates activationand/or proliferation of lymphocytes, 4) modulates the immune response ofan organism, e.g., a mammalian organism, such as a mouse or human, and5) modulates hypoxia associated angiogenesis. See, for example, Toscanoet al. (2007) Cyt Growth Fact Rev 18:57-71; Camby et al. (2006)Glycobiol 16:137 R-157R, each of which is incorporated herein, byreference, in its entirety.

The isolated nucleic acid molecules of the present invention can beused, for example, to express a Gal1 polypeptide or a fragment thereof(e.g., via a recombinant expression vector in a host cell in genetherapy applications), to detect Gal1 mRNA or a fragment thereof (e.g.,in a biological sample) or a genetic alteration in a Gal1 gene, and tomodulate Gal1 activity, as described further below. The Gal1polypeptides or fragments thereof can be used to treat viral-associatedPTLD, e.g., EBV-associated PTLD, and/or hypoxia associated angiogenesisdisorders.

In addition, the Gal1 polypeptides or fragments thereof can be used toscreen for naturally occurring Gal1 binding partner(s), to screen fordrugs or compounds which modulate Gal1 activity, as well as to treathypoxia associated angiogenesis disorders and/or viral-associated PTLD,e.g., EBV-associated PTLD, characterized by insufficient or excessiveproduction of Gal1 polypeptide or a fragment thereof or production ofGal1 polypeptide forms which have decreased, aberrant or unwantedactivity compared to Gal1 wild-type polypeptides or fragments thereof(e.g., viral-associated PTLD, e.g., EBV-associated PTLD). Moreover, theanti-Gal1 antibodies or fragments thereof of the present invention canbe used to detect and isolate Gal1 polypeptides or fragments thereof,regulate the bioavailability of Gal1 polypeptides or fragments thereof,and modulate Gal1 activity, e.g., by modulating the interaction betweena Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof.

A. Screening Assays

In one aspect, the invention relates to a method for preventing in asubject, a disease or condition associated with an unwanted or less thandesirable immune response. Subjects at risk for a disease that wouldbenefit from treatment with the claimed agents and/or methods can beidentified, for example, by any of a combination of diagnostic orprognostic assays known in the art and described herein (see, forexample, agents and assays described in IV. Methods of Selecting Agentsthat Modulate Immune Cell Activation).

B. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining Gal1 polypeptideand/or nucleic acid expression as well as Gal1 activity, in the contextof a biological sample (e.g., blood, serum, cells, or tissue) to therebydetermine whether an individual is afflicted with a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, or is at risk of developing a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, associated with aberrant or unwanted Gal1expression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a hypoxia associated angiogenesis disorder and/or aviral-associated PTLD, e.g., EBV-associated PTLD, associated with Gal1polypeptide, nucleic acid expression or activity. For example, mutationsin a Gal1 gene can be assayed in a biological sample.

Such assays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a hypoxiaassociated angiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, characterized by or associated with Gal1polypeptide, nucleic acid expression or activity.

Another aspect of the present invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds) on the expression oractivity of Gal1 in clinical trials. These and other agents aredescribed in further detail in the following sections.

1. Diagnostic Assays

The present invention provides, in part, methods, systems, and code foraccurately classifying whether a biological sample is associated with ahypoxia associated angiogenesis disorder and/or a viral-associated PTLD,e.g., EBV-associated PTLD, associated with aberrant expression oractivity of by Gal1. In some embodiments, the present invention isuseful for classifying a sample (e.g., from a subject) as associatedwith or at risk for a hypoxia associated angiogenesis disorder and/or aviral-associated PTLD, e.g., EBV-associated PTLD, mediated by Gal1(known as a GAL1 sample and/or Gal1 sample) using a statisticalalgorithm and/or empirical data (e.g., the presence or level of anGal1).

An exemplary method for detecting the level of expression or activity ofGal1 or fragments thereof, and thus useful for classifying whether asample is associated with a disease or disorder mediated by Gal1 or aclinical subtype thereof involves obtaining a biological sample from atest subject and contacting the biological sample with an antibody orantigen-binding fragment thereof of the present invention capable ofdetecting Gal1 such that the level of expression or activity of Gal1 isdetected in the biological sample. In some embodiments, at least oneantibody or antigen-binding fragment thereof is used, wherein two,three, four, five, six, seven, eight, nine, ten, or more such antibodiesor antibody fragments can be used in combination (e.g., in sandwichELISAs) or in serial. In certain instances, the statistical algorithm isa single learning statistical classifier system. For example, a singlelearning statistical classifier system can be used to classify a sampleas a GAL1 sample based upon a prediction or probability value and thepresence or level of Gal1. The use of a single learning statisticalclassifier system typically classifies the sample as a Gal1 sample(e.g., ulcerative colitis) sample with a sensitivity, specificity,positive predictive value, negative predictive value, and/or overallaccuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%.

Other suitable statistical algorithms are well known to those of skillin the art. For example, learning statistical classifier systems includea machine learning algorithmic technique capable of adapting to complexdata sets (e.g., panel of markers of interest) and making decisionsbased upon such data sets. In some embodiments, a single learningstatistical classifier system such as a classification tree (e.g.,random forest) is used. In other embodiments, a combination of 2, 3, 4,5, 6, 7, 8, 9, 10, or more learning statistical classifier systems areused, preferably in tandem. Examples of learning statistical classifiersystems include, but are not limited to, those using inductive learning(e.g., decision/classification trees such as random forests,classification and regression trees (C&RT), boosted trees, etc.),Probably Approximately Correct (PAC) learning, connectionist learning(e.g., neural networks (NN), artificial neural networks (ANN), neurofuzzy networks (NFN), network structures, perceptrons such asmulti-layer perceptrons, multi-layer feed-forward networks, applicationsof neural networks, Bayesian learning in belief networks, etc.),reinforcement learning (e.g., passive learning in a known environmentsuch as naive learning, adaptive dynamic learning, and temporaldifference learning, passive learning in an unknown environment, activelearning in an unknown environment, learning action-value functions,applications of reinforcement learning, etc.), and genetic algorithmsand evolutionary programming. Other learning statistical classifiersystems include support vector machines (e.g., Kernel methods),multivariate adaptive regression splines (MARS), Levenberg-Marquardtalgorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradientdescent algorithms, and learning vector quantization (LVQ). In certainembodiments, the method of the present invention further comprisessending the Gal1 sample classification results to a clinician, e.g., agastroenterologist or a general practitioner.

In another embodiment, the method of the present invention furtherprovides a diagnosis in the form of a probability that the individualhas a condition or disorder associated with aberrant expression oractivity of Gal1. For example, the individual can have about a 0%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or greater probability of having the condition ordisorder. In yet another embodiment, the method of the present inventionfurther provides a prognosis of the condition or disorder in theindividual. In some instances, the method of classifying a sample as aGal1 sample is further based on the symptoms (e.g., clinical factors) ofthe individual from which the sample is obtained. The symptoms or groupof symptoms can be, for example, diarrhea, abdominal pain, cramping,fever, anemia, weight loss, anxiety, depression, and combinationsthereof. In some embodiments, the diagnosis of an individual as having acondition or disorder associated with aberrant expression or activity ofGal1 is followed by administering to the individual a therapeuticallyeffective amount of a drug useful for treating one or more symptomsassociated with the condition or disorder (e.g., chemotherapeuticagents).

In one embodiment, the methods further involve obtaining a controlbiological sample (e.g., biological sample from a subject who does nothave a condition or disorder mediated by Gal1), a biological sample fromthe subject during remission or before developing a condition ordisorder mediated by Gal1, or a biological sample from the subjectduring treatment for developing a condition or disorder mediated byGal1.

An exemplary method for detecting the presence or absence of Gal1polypeptide or nucleic acid or fragments thereof in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting Gal1 polypeptide or nucleic acid that encodes Gal1 polypeptide(e.g., mRNA or genomic DNA) or fragments thereof such that the presenceof Gal1 polypeptide or nucleic acid or fragments thereof is detected inthe biological sample. A preferred agent for detecting Gal1 mRNA,genomic DNA, or fragments thereof is a labeled nucleic acid probecapable of hybridizing to Gal1 mRNA, genomic DNA, or fragments thereof.The nucleic acid probe can be, for example, full length Gal1 nucleicacid, or a portion thereof, such as an oligonucleotide of at least 15,30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to Gal1 mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe present invention are described herein.

A preferred agent for detecting a Gal1 polypeptide or a fragment thereofis an antibody capable of binding to a Gal1 polypeptide, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)2) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin. The term “biological sample” isintended to include tissues, cells, and biological fluids isolated froma subject, as well as tissues, cells, and fluids present within asubject. That is, the detection method of the present invention can beused to detect Gal1 mRNA, polypeptide, genomic DNA, or fragmentsthereof, in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of Gal1 mRNA or a fragmentthereof include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of Gal1 polypeptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of Gal1 genomicDNA or a fragment thereof include Southern hybridizations. Furthermore,in vivo techniques for detection of a Gal1 polypeptide or a fragmentthereof include introducing into a subject a labeled anti-Gal1 antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In one embodiment, the biological sample contains polypeptide moleculesfrom the test subject. Alternatively, the biological sample can containmRNA molecules from the test subject or genomic DNA molecules from thetest subject. A preferred biological sample is a serum sample isolatedby conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting Gal1 polypeptide, mRNA,genomic DNA, or fragments thereof, such that the presence of Gal1polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in thebiological sample, and comparing the presence of Gal1 polypeptide, mRNA,genomic DNA, or fragments thereof, in the control sample with thepresence of Gal1 polypeptide, mRNA, genomic DNA, or fragments thereof inthe test sample.

In still other embodiments, the antibodies can be associated with acomponent or device for the use of the antibodies in an ELISA or RIA.Non-limiting examples include antibodies immobilized on solid surfacesfor use in these assays (e.g., linked and/or conjugated to a detectablelabel based on light or radiation emission as described above). In otherembodiments, the antibodies are associated with a device or strip fordetection of Gal1 by use of an immunochromatographic or immunochemicalassay such as in a “sandwich” or competitive assay. Additional examplesof such devices or strips are those designed for home testing or rapidpoint of care testing. Further examples include those that are designedfor the simultaneous analysis of multiple analytes in a single sample.For example, an unlabeled antibody of the invention may be applied to a“capture” Gal1 polypeptides in a biological sample and the captured (orimmobilized) Gal1 polypeptides may be bound to a labeled form of ananti-Gal1 antibody of the invention for detection. Other standardembodiments of immunoassays are well known the skilled artisan,including assays based on, for example, immunodiffusion,immunoelectrophoresis, immunohistopathology, immunohistochemistry, andhistopathology.

The invention also encompasses kits for detecting the presence of a Gal1nucleic acid, polypeptide, or fragments thereof, in a biological sample.For example, the kit can comprise a labeled compound or agent capable ofdetecting a Gal1 nucleic acid, polypeptide, or fragments thereof in abiological sample; means for determining the amount of the Gal1 nucleicacid, polypeptide, or fragments thereof in the sample; and means forcomparing the amount of the Gal1 nucleic acid, polypeptide, or fragmentsthereof in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect the Gal1 nucleic acid,polypeptide, or fragments thereof.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, associated with aberrant or unwanted Gal1expression or activity. As used herein, the term “aberrant” includes aGal1 expression or activity which deviates from the wild type Gal1expression or activity. Aberrant expression or activity includesincreased or decreased expression or activity, as well as expression oractivity which does not follow the wild type developmental pattern ofexpression or the subcellular pattern of expression. For example,aberrant Gal1 expression or activity is intended to include the cases inwhich a mutation in the Gal1 gene or regulatory sequence thereof causesthe Gal1 gene to be under-expressed or over-expressed and situations inwhich such mutations result in a non-functional Gal1 polypeptide or apolypeptide which does not function in a wild-type fashion, e.g., apolypeptide which does not interact with a Gal1 binding partner(s) orone which interacts with a non-Gal1 binding partner(s). As used herein,the term “unwanted” includes an unwanted phenomenon involved in abiological response such as immune cell activation. For example, theterm unwanted includes a Gal1 expression or activity which isundesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a hypoxia associated angiogenesis disorder and/or aviral-associated PTLD, e.g., EBV-associated PTLD, associated with amisregulation in Gal1 polypeptide activity or nucleic acid expression.Thus, the present invention provides a method for identifying a hypoxiaassociated angiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, associated with aberrant or unwanted Gal1expression or activity in which a test sample is obtained from a subjectand Gal1 polypeptide or nucleic acid (e.g., mRNA or genomic DNA) isdetected, wherein the presence of Gal1 polypeptide or nucleic acid isdiagnostic for a subject having or at risk of developing a hypoxiaassociated angiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, associated with aberrant or unwanted Gal1expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., cerebrospinal fluid orserum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, associated with aberrant or unwanted Gal1expression or activity. For example, such methods can be used todetermine whether a subject can be effectively treated with an agent fora hypoxia associated angiogenesis disorder and/or a viral-associatedPTLD, e.g., EBV-associated PTLD. Thus, the present invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a hypoxia associated angiogenesis disorder and/or aviral-associated PTLD, e.g., EBV-associated PTLD, associated withaberrant or unwanted Gal1 expression or activity in which a test sampleis obtained and Gal1 polypeptide or nucleic acid expression or activityis detected (e.g., wherein the abundance of Gal1 polypeptide or nucleicacid expression or activity is diagnostic for a subject that can beadministered the agent to treat a hypoxia associated angiogenesisdisorder and/or a viral-associated PTLD, e.g., EBV-associated PTLD,associated with aberrant or unwanted Gal1 expression or activity).

The methods of the present invention can also be used to detect geneticalterations in a Gal1 gene, thereby determining if a subject with thealtered gene is at risk for a hypoxia associated angiogenesis disorderand/or a viral-associated PTLD, e.g., EBV-associated PTLD, characterizedby misregulation in Gal1 polypeptide activity or nucleic acidexpression. In preferred embodiments, the methods include detecting, ina sample of cells from the subject, the presence or absence of a geneticalteration characterized by at least one alteration affecting theintegrity of a gene encoding a Gal1 polypeptide, or the mis-expressionof the Gal1 gene. For example, such genetic alterations can be detectedby ascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a Gal1 gene, 2) an addition of one or morenucleotides to a Gal1 gene, 3) a substitution of one or more nucleotidesof a Gal1 gene, 4) a chromosomal rearrangement of a Gal1 gene, 5) analteration in the level of a messenger RNA transcript of a Gal1 gene, 6)aberrant modification of a Gal1 gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a Gal1 gene, 8) a non-wild type levelof a Gal1 polypeptide, 9) allelic loss of a Gal1 gene, and 10)inappropriate post-translational modification of a Gal1 polypeptide. Asdescribed herein, there are a large number of assays known in the artwhich can be used for detecting alterations in a Gal1 gene. A preferredbiological sample is a tissue or serum sample isolated by conventionalmeans from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a Gal1 gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a Gal1 gene under conditions such thathybridization and amplification of the Gal1 gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a Gal1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in Gal1 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotideprobes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J.et al. (1996) Nat. Med. 2:753-759). For example, genetic mutations inGal1 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. (1996) supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequential,overlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Gal1 gene anddetect mutations by comparing the sequence of the sample Gal1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc.Natl. Acad. Sci. USA 74:5463. It is also contemplated that any of avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Naeve, C. W. (1995) Biotechniques19:448-53), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the Gal1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type Gal1 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digest the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Gal1 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Gal1 sequence,e.g., a wild-type Gal1 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility may beused to identify mutations in Gal1 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control Gal1nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to ensure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Gal1 gene.

Furthermore, any cell type or tissue in which Gal1 is expressed may beutilized in the prognostic assays described herein.

Another aspect of the present invention includes uses of thecompositions and methods described herein for association and/orstratification analyses in which the expression level and/or activity ofGal1 in biological samples from individuals with a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, are analyzed and the information is compared tothat of controls (e.g., individuals who do not have the disorder;controls may be also referred to as “healthy” or “normal” individuals orat early timepoints in a given time lapse study) who are preferably ofsimilar age and race. The appropriate selection of patients and controlsis important to the success of association and/or stratificationstudies. Therefore, a pool of individuals with well-characterizedphenotypes is extremely desirable. Criteria for disease diagnosis,disease predisposition screening, disease prognosis, determining drugresponsiveness (pharmacogenomics), drug toxicity screening, etc. aredescribed herein.

Different study designs may be used for genetic association and/orstratification studies (Modern Epidemiology, Lippincott Williams &Wilkins (1998), 609-622). Observational studies are most frequentlycarried out in which the response of the patients is not interferedwith. The first type of observational study identifies a sample ofpersons in whom the suspected cause of the disease is present andanother sample of persons in whom the suspected cause is absent, andthen the frequency of development of disease in the two samples iscompared. These sampled populations are called cohorts, and the study isa prospective study. The other type of observational study iscase-control or a retrospective study. In typical case-control studies,samples are collected from individuals with the phenotype of interest(cases) such as certain manifestations of a disease, and fromindividuals without the phenotype (controls) in a population (targetpopulation) that conclusions are to be drawn from. Then the possiblecauses of the disease are investigated retrospectively. As the time andcosts of collecting samples in case-control studies are considerablyless than those for prospective studies, case-control studies are themore commonly used study design in genetic association studies, at leastduring the exploration and discovery stage.

After all relevant phenotypic and/or genotypic information has beenobtained, statistical analyses are carried out to determine if there isany significant correlation between the presence of an allele or agenotype with the phenotypic characteristics of an individual.Preferably, data inspection and cleaning are first performed beforecarrying out statistical tests for genetic association. Epidemiologicaland clinical data of the samples can be summarized by descriptivestatistics with tables and graphs well known in the art. Data validationis preferably performed to check for data completion, inconsistententries, and outliers. Chi-squared tests and t-tests (Wilcoxon rank-sumtests if distributions are not normal) may then be used to check forsignificant differences between cases and controls for discrete andcontinuous variables, respectively.

An important decision in the performance of genetic association tests isthe determination of the significance level at which significantassociation can be declared when the p-value of the tests reaches thatlevel. In an exploratory analysis where positive hits will be followedup in subsequent confirmatory testing, an unadjusted p-value <0.2 (asignificance level on the lenient side), for example, may be used forgenerating hypotheses for significant association of a SNP with certainphenotypic characteristics of a disease. It is preferred that a p-value<0.05 (a significance level traditionally used in the art) is achievedin order for a SNP to be considered to have an association with adisease. When hits are followed up in confirmatory analyses in moresamples of the same source or in different samples from differentsources, adjustment for multiple testing will be performed as to avoidexcess number of hits while maintaining the experiment-wise error ratesat 0.05. While there are different methods to adjust for multipletesting to control for different kinds of error rates, a commonly usedbut rather conservative method is Bonferroni correction to control theexperiment-wise or family-wise error rate (Multiple comparisons andmultiple tests, Westfall et al, SAS Institute (1999)). Permutation teststo control for the false discovery rates, FDR, can be more powerful(Benjamini and Hochberg, Journal of the Royal Statistical Society,Series B 57, 1289-1300, 1995, Resampling-based Multiple Testing,Westfall and Young, Wiley (1993)). Such methods to control formultiplicity would be preferred when the tests are dependent andcontrolling for false discovery rates is sufficient as opposed tocontrolling for the experiment-wise error rates.

Once individual risk factors, genetic or non-genetic, have been foundfor the predisposition to disease, a classification/prediction schemecan be set up to predict the category (for instance, disease orno-disease) that an individual will be in depending on his phenotypeand/or genotype and other non-genetic risk factors. Logistic regressionfor discrete trait and linear regression for continuous trait arestandard techniques for such tasks (Applied Regression Analysis, Draperand Smith, Wiley (1998)). Moreover, other techniques can also be usedfor setting up classification. Such techniques include, but are notlimited to, MART, CART, neural network, and discriminant analyses thatare suitable for use in comparing the performance of different methods(The Elements of Statistical Learning, Hastie, Tibshirani & Friedman,Springer (2002)).

In addition, the present invention also encompasses kits for detectingthe presence of a Gal1 nucleic acid, polypeptide, or fragments thereof,in a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting a Gal1 nucleic acid, polypeptide,or fragments thereof in a biological sample; means for determining theamount of the Gal1 nucleic acid, polypeptide, or fragments thereof inthe sample; and means for comparing the amount of the Gal1 nucleic acid,polypeptide, or fragments thereof in the sample with a standard. Thecompound or agent can be packaged in a suitable container.

A kit of the present invention may also include instructional materialsdisclosing or describing the use of the kit or an antibody of thedisclosed invention in a method of the disclosed invention as providedherein. A kit may also include additional components to facilitate theparticular application for which the kit is designed. For example, a kitmay additionally contain means of detecting the label (e.g., enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-mouse-HRP,etc.) and reagents necessary for controls (e.g., control biologicalsamples or Gal1 protein standards). A kit may additionally includebuffers and other reagents recognized for use in a method of thedisclosed invention. Non-limiting examples include agents to reducenon-specific binding, such as a carrier protein or a detergent.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., compounds, drugs or smallmolecules) on the expression or activity of a Gal1 polypeptide or afragment thereof (e.g., the modulation of cell proliferation and/ormigration) can be applied not only in basic drug screening, but also inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase Gal1 geneexpression, polypeptide levels, or upregulate Gal1 activity, can bemonitored in clinical trials of subjects exhibiting decreased Gal1 geneexpression, polypeptide levels, or downregulated Gal1 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease Gal1 gene expression, polypeptide levels, ordownregulate Gal1 activity, can be monitored in clinical trials ofsubjects exhibiting increased Gal1 gene expression, polypeptide levels,or Gal1 activity. In such clinical trials, the expression or activity ofa Gal1 gene, and preferably, other genes that have been implicated in,for example, a hypoxia associated angiogenesis disorder and/or aviral-associated PTLD, e.g., EBV-associated PTLD, can be used as a “readout” or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including Gal1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates Gal1 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on a hypoxia associated angiogenesis disorderand/or a viral-associated PTLD, e.g., EBV-associated PTLD, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of Gal1 and other genes implicated in thehypoxia associated angiogenesis disorder and/or viral-associated PTLD,e.g., EBV-associated PTLD, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of polypeptide produced, by one of the methods as describedherein, or by measuring the levels of activity of Gal1 or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide,nucleic acid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a Gal1 polypeptide,mRNA, genomic DNA, or fragments thereof in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the Gal1polypeptide, mRNA, genomic DNA, or fragments thereof in thepost-administration samples; (v) comparing the level of expression oractivity of the Gal1 polypeptide, mRNA, genomic DNA, or fragmentsthereof in the pre-administration sample with the Gal1 polypeptide,mRNA, or genomic DNA in the post administration sample or samples; and(vi) altering the administration of the agent to the subjectaccordingly. For example, increased administration of the agent may bedesirable to increase the expression or activity of Gal1 to higherlevels than detected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of Gal1 to lower levels than detected,i.e., to decrease the effectiveness of the agent. According to such anembodiment, Gal1 expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

D. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a hypoxiaassociated angiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, characterized by insufficient or excessiveproduction of Gal1 polypeptides or production of Gal1 protein formswhich have decreased or aberrant activity compared to Gal1 wild typeprotein. Moreover, the anti-Gal1 antibodies of the present invention canbe used to detect and isolate Gal1 polypeptides or fragments thereof,regulate the bioavailability of Gal1 polypeptides or fragments thereof,and modulate Gal1 activity e.g., by modulating the interaction of a Gal1polypeptide or a fragment thereof with its natural binding partner(s) orfragments(s) thereof.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedGal1 expression or activity, by administering to the subject a Gal1polypeptide or a fragment thereof or an agent which modulates Gal1expression or at least one Gal1 activity. Subjects at risk for a hypoxiaassociated angiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD associated with aberrant or unwanted Gal1 expressionor activity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the Gal1 aberrancy, such that a hypoxia associatedangiogenesis disorder and/or a viral-associated PTLD, e.g.,EBV-associated PTLD, is prevented or, alternatively, delayed in itsprogression. Depending on the type of Gal1 aberrancy, for example, aGal1 polypeptide, Gal1 agonist or Gal1 antagonist (e.g., an anti-Gal1antibody or a combination of anti-Gal1 and antibodies against otherimmune related targets) agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

Another aspect of the present invention pertains to methods ofmodulating Gal1 expression or activity or interaction with its naturalbinding partner(s), for therapeutic purposes. The activity and/orexpression of Gal1, as well as the interaction between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof can be modulated in order to modulate the immuneresponse.

Modulatory methods of the present invention involve contacting a cellwith a Gal1 polypeptide or a fragment thereof or agent that modulatesone or more of the activities of Gal1 polypeptide activity associatedwith the cell, e.g., an agent that modulates expression or activity ofGal1 and/or modulates the interaction of a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof. An agent that modulates Gal1 polypeptide activity can be anagent as described herein, such as a nucleic acid or a polypeptide, anaturally-occurring binding partner of a Gal1 polypeptide, a Gal1antibody, a combination of Gal1 antibodies and antibodies against otherimmune related targets, a Gal1 agonist or antagonist, a peptidomimeticof a Gal1 agonist or antagonist, a Gal1 peptidomimetic, other smallmolecule, or small RNA directed against a Gal1 nucleic acid geneexpression product.

An agent that modulates the expression of Gal1 is, e.g., an antisensenucleic acid molecule, RNAi molecule, shRNA or other small RNA molecule,triplex oligonucleotide, ribozyme, or recombinant vector for expressionof a Gal1 polypeptide. For example, an oligonucleotide complementary tothe area around a Gal1 polypeptide translation initiation site can besynthesized. One or more antisense oligonucleotides can be added to cellmedia, typically at 200 μg/ml, or administered to a patient to preventthe synthesis of a Gal1 polypeptide. The antisense oligonucleotide istaken up by cells and hybridizes to a Gal1 mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of Gal1 polypeptide isblocked. When Gal1 expression is modulated, preferably, such modulationoccurs by a means other than by knocking out the Gal1 gene.

Agents which modulate expression, by virtue of the fact that theycontrol the amount of Gal1 in a cell, also modulate the total amount ofGal1 activity in a cell.

In one embodiment, the agent the modulates Gal1 stimulates one or moreGal1 activities. Examples of such stimulatory agents include active Gal1polypeptide or a fragment thereof and a nucleic acid molecule encodingGal1 or a fragment thereof that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more Gal1 activities. In apreferred embodiment, the agent inhibits or enhances the interaction ofGal1 with its natural binding partner(s). Examples of such inhibitoryagents include antisense Gal1 nucleic acid molecules, anti-Gal1antibodies, Gal1 inhibitors, and compounds identified in the subjectscreening assays.

These modulatory methods can be performed in vitro (e.g., by contactingthe cell with the agent) or, alternatively, by contacting an agent withcells in vivo (e.g., by administering the agent to a subject). As such,the present invention provides methods of treating an individualafflicted with a viral-associated PTLD, e.g., EBV-associated PTLD, thatwould benefit from up- or down-modulation of a Gal1 polypeptide or afragment thereof. In one embodiment, the method involves administeringan agent (e.g., an agent identified by a screening assay describedherein), or combination of agents that modulates (e.g., upregulates ordownregulates) Gal1 expression or activity. In another embodiment, themethod involves administering a Gal1 polypeptide or nucleic acidmolecule as therapy to compensate for reduced, aberrant, or unwantedGal1 expression or activity.

Stimulation of Gal1 activity is desirable in situations in which Gal1 isabnormally downregulated and/or in which increased Gal1 activity islikely to have a beneficial effect. Likewise, inhibition of Gal1activity is desirable in situations in which Gal1 is abnormallyupregulated and/or in which decreased Gal1 activity is likely to have abeneficial effect.

Exemplary agents for use in downmodulating Gal1 (i.e., Gal1 antagonists)include, e.g., antisense nucleic acid molecules, antibodies thatrecognize and block Gal1, combinations of antibodies that recognize andblock Gal1 and antibodies that recognize and block other immune relatedtargets, and compounds that block the interaction of a Gal1 polypeptideor a fragment thereof with its naturally occurring binding partner(s) orfragment(s) thereof on an immune cell. Exemplary agents for use inupmodulating Gal1 (i.e., Gal1 agonists) include, e.g., nucleic acidmolecules encoding Gal1 polypeptides, multivalent forms of Gal1,compounds that increase the expression of Gal1, compounds that enhancethe interaction of Gal1 with its naturally occurring binding partner(s)and cells that express Gal1.

In addition, these modulatory agents can also be administered incombination therapy with, e.g., chemotherapeutic agents, hormones,antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy,and/or radiotherapy. The preceding treatment methods can be administeredin conjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy. For example,these modulatory agents can be administered with a therapeuticallyeffective dose of chemotherapeutic agent. In another embodiment, thesemodulatory agents are administered in conjunction with chemotherapy toenhance the activity and efficacy of the chemotherapeutic agent. ThePhysicians' Desk Reference (PDR) discloses dosages of chemotherapeuticagents that have been used in the treatment of various cancers. Thedosing regiment and dosages of these aforementioned chemotherapeuticdrugs that are therapeutically effective will depend on the particularviral-associated PTLD, e.g., EBV-associated PTLD, being treated, theextent of the disease and other factors familiar to the physician ofskill in the art and can be determined by the physician.

3. Upregulation of Immune Responses and/or Downregulation of HypoxiaAssociated Angiogenesis

Also useful therapeutically is the inhibition of interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof to thereby upregulate immuneresponses and/or downregulate hypoxia associated angiogenesis.Upregulation of immune responses and/or downregulation of hypoxiaassociated angiogenesis can be in the form of enhancing an existing oreliciting an initial immune response and/or anti-hypoxia associatedangiogenesis response. In one embodiment, an agent that blocksinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof is used to enhancethe immune response and/or downregulate hypoxia associated angiogenesis.Such an agent (e.g., a Gal1 blocking antibody) is therapeutically usefulin situations where upregulation of antibody and cell-mediated responseswould be beneficial.

Alternatively, immune responses and/or anti-hypoxia associatedangiogenesis can be enhanced in an infected patient through an ex vivoapproach, for instance, by removing cells, such as immune cells, fromthe patient, contacting immune cells in vitro with an agent that blocksinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof, and reintroducingthe in vitro stimulated immune cells into the patient.

In certain instances, it may be desirable to further administer otheragents that upregulate immune responses, for example, forms of B7 familymembers that transduce signals via costimulatory receptors, in order tofurther augment the immune response. In other embodiments, suchadditional agents can comprise anti-angiogenesis agents such asanti-VEGF therapies well known in the art.

An agent that inhibits Gal1 activity or interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof, can be used prophylactically in vaccines againstvarious polypeptides, e.g., polypeptides derived from pathogens.Immunity against a pathogen, e.g., a virus, can be induced byvaccinating with a viral polypeptide along with an agent that inhibitsGal1 activity or interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof, inan appropriate adjuvant. Alternately, a vector comprising genes whichencode for both a pathogenic antigen and a form of Gal1 that blocksinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof can be used forvaccination. Nucleic acid vaccines can be administered by a variety ofmeans, for example, by injection (e.g., intramuscular, intradermal, orthe biolistic injection of DNA-coated gold particles into the epidermiswith a gene gun that uses a particle accelerator or a compressed gas toinject the particles into the skin (Haynes et al. (1996) J. Biotechnol.44:37)). Alternatively, nucleic acid vaccines can be administered bynon-invasive means. For example, pure or lipid-formulated DNA can bedelivered to the respiratory system or targeted elsewhere, e.g., Peyerspatches by oral delivery of DNA (Schubbert (1997) Proc. Natl. Acad. Sci.USA 94:961). Attenuated microorganisms can be used for delivery tomucosal surfaces (Sizemore et al. (1995) Science 270:29).

In another embodiment, the antigen in the vaccine is a self-antigen.Such a vaccine is useful in the modulation of tolerance in an organism.Immunization with a self antigen and an agent that blocks Gal1 activityor interactions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof can break tolerance(i.e., interfere with tolerance of a self antigen). Such a vaccine mayalso include adjuvants such as alum or cytokines (e.g., GM-CSF, IL-12,B7-1, or B7-2).

In another embodiment, upregulation or enhancement of an immune responsefunction and/or downregulation of hypoxia associated angiogenesis, asdescribed herein, is useful in the induction of tumor immunity (e.g.,restoration of immune surveillance in viral-associated PTLD, e.g.,EBV-associated PTLD). Viral-associated PTLD cells can be transfectedwith a nucleic acid molecule that inhibits Gal1 activity or interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof. These molecules can be, e.g.,nucleic acid molecules which are antisense to Gal1, or can encodenon-activating anti-Gal1 antibodies or combinations of anti-Gal1antibodies and antibodies against other immune related targets. Thesemolecules can also be the variable region of an anti-Gal1 antibodyand/or an anti-Gal1 antibody. If desired, the tumor cells can also betransfected with other polypeptides which enhance an immune response.The transfected tumor cells are returned to the patient, which resultsin inhibition (e.g., local inhibition) of Gal1 activity or interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof. Alternatively, gene therapytechniques can be used to target a tumor cell for transfection in vivo.

Stimulation of an immune response to tumor cells and/or downregulationof hypoxia associated angiogenesis can also be achieved by inhibitingGal1 activity or interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof, bytreating a patient with an agent that inhibits Gal1 activity orinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. Examples of suchagents include, e.g., antisense nucleic acid molecules, small RNAs,antibodies that recognize and block Gal1, a combination of antibodiesthat recognize and block Gal1 and antibodies that recognize and blockother immune- and/or angiogenesis-related targets, compounds that blockthe interactions between a Gal1 polypeptide or a fragment thereof andits natural binding partner(s) or a fragment(s) thereof on an immunecell, and compounds identified in the subject screening assays).

In another embodiment, the immune response can be stimulated by themethods described herein, such that preexisting tolerance and/orimmunosuppression is overcome. For example, immune responses againstantigens to which a subject cannot mount a significant immune response,e.g., to an autologous antigen, such as a tumor specific antigens can beinduced by administering an agent that blocks interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof.

In one embodiment, a blocking antibody that inhibits interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof can be used to enhance an immuneresponse (e.g., to a tumor cell). In one embodiment, an autologousantigen, such as a tumor-specific antigen can be coadministered. Inanother embodiment, an immune response can be stimulated against anantigen (e.g., an autologous antigen) to treat a viral-associated PTLD,e.g., EBV-associated PTLD. In another embodiment, the subject agents canbe used as adjuvants to boost responses to foreign antigens in theprocess of active immunization.

In one embodiment, immune cells are obtained from a subject and culturedex vivo in the presence of an agent as described herein, to expand thepopulation of immune cells and/or to enhance immune cell activation. Ina further embodiment the immune cells are then administered to asubject. Immune cells can be stimulated in vitro by, for example,providing to the immune cells a primary activation signal and acostimulatory signal, as is known in the art. Various agents can also beused to costimulate proliferation of immune cells. In one embodimentimmune cells are cultured ex vivo according to the method described inPCT Application No. WO 94/29436. The costimulatory polypeptide can besoluble, attached to a cell membrane, or attached to a solid surface,such as a bead.

In an additional embodiment, in performing any of the methods describedherein, it is within the scope of the present invention to upregulate animmune response by administering one or more additional agents. Forexample, the use of other agents known to stimulate the immune response,such as cytokines, adjuvants, or stimulatory forms of costimulatorymolecules or their ligands can be used in conjunction with an agent thatinhibits Gal1 activity or a Gal1 polypeptide or a fragment thereof andits natural binding partner(s) or a fragment(s) thereof.

V. Administration of Agents

The immune modulating agents of the present invention are administeredto subjects in a biologically compatible form suitable forpharmaceutical administration in vivo, to either enhance or suppressimmune cell mediated immune responses. By “biologically compatible formsuitable for administration in vivo” is meant a form of the protein tobe administered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term “subject” is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Administration of an agent asdescribed herein can be in any pharmacological form including atherapeutically active amount of an agent alone or in combination with apharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeuticcomposition of the present invention is defined as an amount effective,at dosages and for periods of time necessary, to achieve the desiredresult. For example, a therapeutically active amount of a Gal1 blockingantibody may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of peptide to elicita desired response in the individual. Dosage regimens can be adjusted toprovide the optimum therapeutic response. For example, several divideddoses can be administered daily or the dose can be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The agents or the invention described herein can be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound can be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. For example, for administration of agents, byother than parenteral administration, it may be desirable to coat theagent with, or co-administer the agent with, a material to prevent itsinactivation.

An agent can be administered to an individual in an appropriate carrier,diluent or adjuvant, co-administered with enzyme inhibitors or in anappropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

The agent may also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof, and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Pharmaceutical compositions of agents suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. In all cases the composition willpreferably be sterile and must be fluid to the extent that easysyringeability exists. It will preferably be stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an agentof the present invention (e.g., an antibody, peptide, fusion protein orsmall molecule) in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe agent plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When the agent is suitably protected, as described above, the proteincan be orally administered, for example, with an inert diluent or anassimilable edible carrier. As used herein “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form”, as used herein, refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the present invention are dictated by, and directly dependenton, (a) the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and (b) the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

In one embodiment, an agent of the present invention is an antibody. Asdefined herein, a therapeutically effective amount of antibody (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an antibody can include a single treatment or,preferably, can include a series of treatments. In a preferred example,a subject is treated with antibody in the range of between about 0.1 to20 mg/kg body weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody used for treatmentmay increase or decrease over the course of a particular treatment.Changes in dosage may result from the results of diagnostic assays. Inaddition, an antibody of the present invention can also be administeredin combination therapy with, e.g., chemotherapeutic agents, hormones,antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy,and/or radiotherapy. An antibody of the present invention can also beadministered in conjunction with other forms of conventional therapy,either consecutively with, pre- or post-conventional therapy. Forexample, the antibody can be administered with a therapeuticallyeffective dose of chemotherapeutic agent. In another embodiment, theantibody can be administered in conjunction with chemotherapy to enhancethe activity and efficacy of the chemotherapeutic agent. The Physicians'Desk Reference (PDR) discloses dosages of chemotherapeutic agents thathave been used in the treatment of various cancers. The dosing regimentand dosages of these aforementioned chemotherapeutic drugs that aretherapeutically effective will depend on the particular viral-associatedPTLD, e.g., EBV-associated PTLD, being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Anti-Gal1 Monoclonal Antibodies

Anti-Gal1 monoclonal antibodies were generated and reacted with humanrecombinant Gal1 and endogenous Gal1 in biochemical assays (FIG. 1) andin immunohistochemical analyses of primary tumors. In addition, severalof the newly developed Gal1 monoclonal antibodies also cross-reactedwell with endogenous Gal1 from cynomologous monkey and mouse (FIG. 2).Epitope mapping indicated that the 8B5, 8F4 and 8G3 Gal1 monoclonalantibodies all recognized a domain distal to the previously describedcarbohydrate-binding domain (FIGS. 3-4 and Table 1).

These antibodies (i.e., 8B5, 8F4, and 8G3) were subsequently sequencedand determined to each have the same sequence, with the light chainbeing lambda. Briefly, total RNA was extracted from each hybridoma andsubjected to RT-PCR using constant region specific 3′ primers and poolsof degenerate signal sequence specific 5′ primers. Amplified productswere cloned and sequenced. For the heavy chain, a total of 36 cloneswere sequenced; and for the light chain, a total of 19 clones weresequenced. Sequence alignments yielded the same heavy and light chainsequences for all clones across all three antibodies. These sequencesare presented in Table 1 below and analysis of the sequences obtainedfrom the hybridomas is summarized in Table 2 below. In addition,hybridoma cell line 14-19 8F4-F8-G7 was deposited with the American TypeCulture Collection having an address of 10801 University Boulevard,Manassas, Va. 20110-2209 U.S.A. and was received on Dec. 17, 2009 inaccordance with the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure under deposit number PTA-10535. Inaccordance with the United States Code Of Federal Regulations (see 37CFR 1.808) and the United States Patent And Trademark Office's Manual OfPatent Examination (“MPEP”) (see §2410.01), all restrictions imposed bythe depositor on the availability to the public of the depositedmaterial (except as permitted by the MPEP) will be irrevocably removedupon the granting of any patent issuing from this application or fromany continuing application based thereon.

TABLE 1Epitope mapping and sequences of anti-human Gal1 monoclonal antibodiesmAbs Mapping Domain recognition 8B5.E6.2H3 GST-F5; GST-F6; GST-F7Post-CBD 8B5.E6.H9 GST-F5; GST-F6; GST-F7 Post-CBD 8F4.F8.G7GST-F5; GST-F6; GST-F7 Post-CBD 8F4.F8.H2 GST-F5; GST-F6; GST-F7Post-CBD 8G3.B1.G12 GST-F5; GST-F6; GST-F7 Post-CBD 8G3.B1.H8GST-F5; GST-F6; GST-F7 Post-CBD 2E5.2H12 GST-F3; GST-F6; GST-F7 CBD28B5, 8F4, and 8G3 Heavy Chain Variable (vH) DNA (SEQ ID NO: 6) and Amino Acid (SEQ ID NO: 7) Sequences*        10        20        30        40        50        60        70        80        90       100GAGGTTCAGCTGCAGCAGTCTGTGGCAGAGTTTGTGAGGCCAGGGGCCTCAGTCAGGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAAACACCTATA E  V  Q  L  Q  Q  S  V  A  E  F  V  R  P  G  A  S  V  R  L  S  C  T  A  S  G  F  N  I  K  N  Y  Y                           10                            20                            30       110       120       130       140       150       160       170       180       190       200TACACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAAGATTGATCCTGCGAATGGTAATACTAAATATTTCCCGGAGTTCCAGGGCAAI  H  W  V  R  Q  R  P  E  Q  G  L  E  W  I  G  K  I  D  P  A  N  G  N  I  N  K  V  P  E  F  Q  G  K                 40                            50    52  a                      60       210       220       230       240       250       260       270       280       290       300GGCCACTATGACTGCGGACACATCCTCCAACACAGTCTACCTGCACCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGTCGATGGTTAC  A  T  N  I  A  D  T  S  S  N  T  V  Y  L  N  L  S  S  L  T  S  E  D  T  A  T  Y  Y  Q  V  D  G  Y          70                            80    82  a  b  c                      90       310       320       330       340       350TACGGCTGGTATTTCGCTGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA Y  G  K  Y  F  A  V  W  G  T  G  T  T  V  T  V  S  S        100  a                           1108B5, 8F4, and 8G3, including 8F4F8G7, Light Chain Variable (vL) DNA (SEQ ID NO: 8) and Amino Acid(SEQ ID NO: 9) Sequences*        10        20        30        40        50        60        70        80        90       100CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGCACTGGGGCTGTTACAACTAGTAACT Q  A  V  V  T  Q  E  S  A  L  T  T  S  P  G  E  T  V  T  L  T  C  R  S  S  T  G  A  V  T  T  S  S                         9 11                         20                   27  a  b  c       30       110       120       130       140       150       160       170       180       190       200ATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGCTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCY  A  N  W  V  Q  E  K  P  D  H  L  F  T  G  L  I  G  A  T  N  N  R  A  P  G  V  P  A  R  F  S  G  S                       40                            50                            60       210       220       230       240       250       260       270       280       290       300CCTGATTGGAGACAAGGCTGTCCTCACCATCACAGGGGCACAAACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGAAACCATTTTATTTTC  L  I  G  D  K  A  V  L  T  I  F  G  A  Q  T  E  D  E  A  I  Y  F  C  A  L  W  Y  R  N  H  F  I  F             70                            80                            90       310       320 GGCAGTGGAACCAAGGTCACTGTCCTC G  S  G  T  K  V  I  V  L   100               106  a8B5, 8F4, and 8G3, including 8F4F8G7, Heavy Chain Variable (vH) DNA Sequence (SEQ ID NO: 6)*GAGGTTCAGCTGCAGCAGTCTGTGGCAGAGTTTGTGAGGCCAGGGGCCTCAGTCAGGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAAACACCTATATACACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAAGATTGATCCTGCGAATGGTAATACTAAATATGTCCCGGAGTTCCAGGGCAAGGCCACTATGACTGCGGACACATCCTCCAACACAGTCTACCTGCACCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGTCGATGGTTACTACGGCTGGTATTTCGCTGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA8B5, 8F4, and 8G3, including 8F4F8G7, Light Chain Variable (vλ) DNA Sequence*CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGCACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGCTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGTCCTCACCATCACAGGGGCACAAACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGAAACCATTTTATTTTCGGCAGTGGAACCAAGGTCACTGTC CTC8B5, 8F4, and 8G3, including 8F4F8G7, Heavy Chain Variable (vH) Amino AcidSequence (SEQ ID NO: 7)*EVQLQQSVAEFVRPGASVRLSCTASGFNIKNTYIHWVRQRPEQGLEWIGKIDPANGNTKYVPEFQGKATMTADTSSNTVYLHLSSLTSEDTAIYYCVDGYYGWYFAVWGT GTTVTVSS8B5, 8F4, and 8G3, including 8F4F8G7, Light Chain Variable (vλ) DNASequence* QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGATNNRAPGVPARFSGSLIGDKAVLTITGAQTEDEAIYFCALWYRNHFIFGSGTKVTVL *CDR definitionsand protein sequence numbering according to Kabat. CDR nucleotide andprotein sequences are highlighted in red color or underlining in orderof CDR1, CDR2, and CDR3, respectively.

TABLE 2 Summary of sequences of anti-human Gal1 monoclonal antibodiesAntibody Sequence Analysis^(a) H Chain L Chain CDR 1 Length 5 aa 14 aaCDR 2 Length 17 aa 7 aa CDR 3 Length 9 aa 9 aa Closest HumanGermline^(b) IGHV1-46 (62%) IGLV7-46 (62%) Closest Human FW1^(b)IGHV3-49 (67%) IGLV7-46 (82%) Closest Human FW2^(b) IGHV1-46 (79%)IGLV7-43 (42%) Closest Human FW3^(b) IGHV1-46 (70%) IGlV7-46 (73%)Closest Human J^(b) IGHJ6 (92%) IGLJ1 (90%) ^(a)CDR definitions andsequence numbering according to Kabat ^(b)Germline ID(s) indicatedfollowed by % homology

Example 2 Materials and Methods for Examples 3-8

A. Cell Lines

The L428 cHL cell line (L428), the SU-DHL6 DLBCL cell line and thirteenEBV-transformed B-lymphoblastoid cell lines (LCLs) (NOR-, RIC-, STA-,FOL-, LOV-, MV-, WOL-, FW-, VS-, MA-, SC-, DS-, AND DW-LCL) weremaintained in RPMI-1640 supplemented with 10% FBS (Cellgro Media Tech,Manassas, Va.), 2 mM glutamine, 50 u/ml penicillin and 50 u/mlstreptomycin. The 293T cell line was purchased from ATCC and maintainedin Dulbecco's Modified Eagle's Medium supplemented with 10% FBS.

B. Analysis of Gal1 Transcript Abundance by Gene Expression Profiling

Gene expression profiling data were obtained for two previouslydescribed data sets (Vockerodt et al. (2008) J Pathol (2008) 216:83-92;Basso et al. (2005) Nature Genetics 37:382-90) from the Gene ExpressionOmnibus (accession numbers GSE2350 and GSE10821) and individuallynormalized by robust multiarray preprocessing. Data from Basso et al.(Basso et al. (2005) Nature Genetics 37:382-90) was utilized forevaluation of Gal-1 expression across a panel of 4 HL cell lines, 5LCLs, 20 normal human B-cell samples and 42 additional B-cell neoplasms.Data from Vockerodt et al. (Vockerodt et al. (2008) J Pathol (2008)216:83-92) was utilized for differential gene expression analysis oftranscriptional changes induced by LMP1. This was performed within thespace of top 10,000 most variable probes in the dataset, as ranked bymedian absolute deviation. Differences in probe intensity betweenLMP1-positive and LMP1-negative samples were assessed with asignal-to-noise ratio metric corrected for multiple hypothesis testingby 10,000 permutations using a previously described method (Storey etal. (2003) Proc Natl Acad Sci USA 100:9440-5).

C. Generation and Characterization of Anti-Human Gal1 MonoclonalAntibodies (mAbs)

Anti-human Gal1 mAbs were obtained by immunizing B6-Cg-Tg (BCL2)22Wehi-Jmice (Jackson Labs, Bar Harbor, Me.) with recombinant humanglutathione-s-transferase (GST)-Gal1, generating anti-Gal1 hybridomaswith standard methods and purifying the Gal1 monoclonal antibody andclass-matched IgG2bλ control by affinity chromatography. The specificityof the Gal1 monoclonal antibody was demonstrated by performingenzyme-linked immunosorbent assay (ELISA) of recombinant GST-Gal1 andHis-Gal1 and immunoblotting recombinant human Gal1 (rGal1) andendogenous Gal1 from HL cell lines. A previously described anti-Gal1polyclonal antibody (Juszczynski et al. (2007) Proc Natl Acad Sci USA104:13134-9) was used as a positive control in all assays.

D. Immunoblotting

Expression of Gal1 protein in HL, LCL and DLBCL cell lines wasdetermined by Western blot (WB) using the αGal1 monoclonal antibody(8F4F8G7) or the previously described polyclonal antiserum (Juszczynskiet al. (2007) Proc Natl Acad Sci USA 104:13134-9). Knock-down of LMP2Awas confirmed by WB using an αLMP2a antibody (Abcam, Cambridge, Mass.).Activity of the AP-1 components, cJun and JunB, in the HL cell line L428and the LCLs RIC and NOR were interrogated by WB usingαphospho(Ser63)-cJun (Cell Signaling Technology), αcJun (Cell SignalingTechnology, Danvers, Mass.), aphospho(Ser259)-JunB (Santa CruzBiotechnology, Inc. Santa Cruz, Calif.) and αJunB (Cell SignalingTechnology). WBs were normalized using αβ-actin antibody (Sigma Aldrich,St. Louis, Mo.) to determine β-actin expression as a loading control.

E. Immunohistochemistry of Primary Tumor Specimens

A series of biopsies of newly diagnosed primary PTLDs and diffuse largeB-cell lymphomas (DLBCLs) were obtained from the Brigham & Women'sHospital (BWH) archives with Institutional Review Board (IRB) approval.Immunohistochemistry (IHC) for Gal1, phospho-cJun and JunB was performedusing 5 μm thick formalin-fixed, paraffin-embedded tissue sections.Antigen retrieval was conducted using a steam pressure cooker and 10 mMcitrate buffer, pH 6.0 (Invitrogen, for JunB and Galectin1) or 1 mMEDTA, pH 8.0 (Invitrogen, for phospho-c-Jun) as described previously(Rodig et al. (2009) Clin Cancer Res 14:3338-44). All further steps wereperformed at room temperature in a hydrated chamber. Slides wereinitially treated with Peroxidase Block (DAKO USA, Carpinteria, Calif.)for 5 min to quench endogenous peroxidase activity and subsequentlyincubated with either αJunB (clone C37F9, 1:1000 dilution, CellSignaling Technology), αc-Jun specific for phosphorylated serine atamino acid 63 (clone 54B3, 1:50 dilution, Cell Signaling Technology),and αgalectin1 (clone 8F4.8F.67, 1:40,000 dilution), or rabbit Gal1antiserum in antibody diluent (DAKO) for 1 h. Thereafter, slides werewashed in 50 mM Tris-Cl, 0.05% Tween 20, pH 7.4 and anti-mouse or rabbithorseradish peroxidase-conjugated antibody (Envision Plus, DAKO) wasapplied for 30 min. After further washing, immunoperoxidase staining wasdeveloped using diaminobenzidine (DAB) chromogen (DAKO) per themanufacturer. Slides were also counterstained with Harris hematoxylin.

F. Generation of LMP1 and LMP2A Constructs and Analysis of Gal1 PromoterConstructs with Luciferase Assays.

Total RNA from EBV-transformed LCLs was obtained using standard methodsand reverse transcribed with Super™Script RT 111 (Invitrogen, Carlsbad,Calif.) and LMP1 and LMP2A gene-specific primers (AAGAAAGGTTAGTCATAG(SEQ ID NO: 10) and TGTAAGGCAGTAGTAG (SEQ ID NO: 11), respectively).LMP1 and LMP2A cDNAs were then PCR-amplified using following primerpairs: LMP1-F: GAAGAATTCGATGGAACACGACCTTGAG (SEQ ID NO: 12); LMP1-R:GACAGATCTAGGTTAG TCATAGTAGCTTAG (SEQ ID NO: 13); LMP2A-F:GAATTCTGCAGCTATGGGGTCCCTA (SEQ ID NO: 14); LMP2A-R:AGATCTGCGATCTGGTGGGCATTCT (SEQ ID NO: 15). PCR products were digestedwith EcoRI and BglII and ligated in the pFLAG-CMV2 vector (SigmaAldrich, St. Louis, Mo.). The control reporter plasmid, pRL-TK, wasmodified by substituting the TK promoter for the phosphoglucokinase(PGK) promoter to avoid LMP1/LMP2A transactivation of the controlreporter in luciferase assays. For luciferase assays, the 293T cell linewas grown to 60-80% confluency on 6 well-plates and co-transfected with150 ng/well of the previously described LGALS1 promoter pGL3 construct(Juszczynski et al. (2007) Proc Natl Acad Sci USA 104:13134-9), 100ng/well of the control reporter plasmid, pRL-PGK, and 150 ng/well ofLMP1-FLAG and/or LMP2A-FLAG or 150-300 ng of empty pFLAG-CMV2 vector(total amount of 550 ng of combined plasmids per well). Transfection wasperformed using FuGENE® 6 transfection reagent (Roche Applied Science,Indianapolis, Ind.) according to manufacturer's protocol. After 24 h ofincubation, cells were lysed and luciferase activities were determinedby chemiluminescence assay using the Dual Luciferase Assay kit (Promega,Madison, Wis.) and Luminoskan Ascent luminometer (Thermo Lab Systems,Franklin, Mass.) as previously described (Juszczynski et al. (2007) ProcNatl Acad Sci USA 104:13134-9).

G. RNA Interference-Mediated LMP2A Depletion

LMP2A siRNA oligos were designed using the Dharmacon siRNA design tool(available at the Dharmacon company website) and LMP2A mRNA (GenBankaccession # Y00835) as a template. Two independent LMP2A siRNA oligos(oligo 1 target sequence: NNACACUUAACUUGACUACAA (SEQ ID NO: 16); oligo 2target sequence: NNACUAGGAACCCAAGAUCAA (SEQ ID NO: 17)) were obtainedfrom Dharmacon (Lafayette, Colo.) and a non-targeting siRNA control (SCRoligo) was obtained from AMBION (Cambridge, Mass.). For siRNAnucleofections, 4×10⁶ of NOR-LCL cells transfected by electroporationusing AMAXA nucleofector solution R containing 75 pmoles of LMP2A or SCRoligo and treated with V-001 program in the Nucleofector II device(AMAXA, Koeln, Germany). Transduction efficiency was confirmed to beabove 90% by nucleofection of Cy3-labeled GAPDH oligo (AppliedBiosystems/Ambion, Austin, Tex.) and subsequent flow cytometry analysis.After nucleofections, NOR cells were incubated for 72 h and whole-cellextracts were subsequently prepared for immunoblotting.

H. Analysis of AP-1 Activity and Binding to Gal1 Enhancer

Chromatin Immunoprecipitation-coupled Polymerase Chain Reaction(ChIP-PCR) was used to analyze the binding of cJun and JunB to the Gal1enhancer region (Juszczynski et al. (2007) Proc Natl Acad Sci USA104:13134-9) in EBV-transformed LCLs and the L428 cHL cell line. Assayswere performed using 4×10⁷ cells and a SIMPLECHIP® EnzymaticImmunoprecipitation Chromatin IP Kit (Cell Signaling Technology)according to the manufacturer's protocol. Chromatin wasimmunoprecipitated with rabbit monoclonal α-cJun (Clone 60A8), α-JunB(Clone C37F9) or control rabbit Ig (all obtained from Cell SignalingTechnologies). Thereafter, chromatin immunoprecipitates were evaluatedfor Gal1 enhancer sequences by PCR using the primers specific for thepreviously described AP-1 dependent Gal1 enhancer (Juszczynski et al.(2007) Proc Natl Acad Sci USA 104:13134-9) and reference to 2% input DNAsamples. PCRs were performed using Phusion Hot Start High Fidelity DNAPolymerase reagents (Finnzyme, Woburn, Mass.) according to themanufacturer's protocol (Primer Sequences: 5′-CCAAGCCCACATCTCCTC-3′ (SEQID NO: 18), 5′-GAGGCTGCAGCTGGTTTAGT-3′ (SEQ ID NO: 19)), amplified for35 cycles and subsequently evaluated by agarose gel electrophoresis.Densitometric analysis of bands was performed using ImageJ software(National Institutes of Health, Bethesda, Md.). Additional assays ofGal1 promoter and enhancer-driven luciferase activity in EBV-transformedLCLs were performed as previously described (Juszczynski et al. (2007)Proc Natl Acad Sci USA 104:13134-9). In brief, NOR cells werecotransfected with 300 ng of the pGL3-Gal1-promoter constructs (withoutor with wild-type or mutant AP-1 dependent enhancer) and 100 ng of thecontrol reporter plasmid, pRL-TK, and evaluated for relative luciferaseactivity as described (Juszczynski et al. (2007) Proc Natl Acad Sci USA104:13134-9). Endogenous levels of total and active c-Jun and JunB wereevaluated by immunoblotting.

I. Inhibition of PI3K and NFκB Activity

NFκB activity was inhibited by overexpressing of an IκBαsuper-repressorconstruct (cloned into MSCV-eGFP backbone) in the EBV-transformed LCL,NOR (Feuerhake et al. (2005) Blood 106:1392-9). SR-IκBα, which cannot bephosphorylated by IκK, remains in complex with the NFκB heterodimer,inhibiting NFκB translocation and activation of NFκB targets. Retroviralsupernatants were generated by co-transfecting MSCV-based SR-IκBα withpKAT and VSV-G vectors into 293T cells as previously described(Juszczynski et al. (2006) Mol Cell Biol 26:5348-59). Supernatantscontaining retrovirus were harvested at 24 hours and used to infectEBV-transformed LCLs as previously described (Juszczynski et al. (2006)Mol Cell Biol 26:5348-59). Seventy-two or 96 hours after infection,eGFP+ cells were sorted using a B-D FACS Aria II sorter and lysates wereprepared for immunoblotting. PI3K/Akt activity was inhibited using aPI3K chemical inhibitor, Ly294002 (Calbiochem). LCLs were treated with25 μM Ly294002 or the equivalent volume of DMSO as a vehicle control for72 hours and lysed thereafter for immunoblotting.

J. Anti-Gal1 mAb-Mediated Neutralization of Recombinant Gal1-InducedT-Cell Apoptosis

Normal T cells were purified and activated with a combination of αCD3(0.1 μg/ml) and αCD28 (0.5 μg/ml) as previously described (Juszczynskiet al. (2007) Proc Natl Acad Sci USA 104:13134-9). Ten μM recombinanthuman Gal1 (rGal1) was pre-incubated with 5 μM anti-Gal1 mAb 8F4F8G7 orisotype control IgG2bλ (Rockland Immunochemicals Inc., Boyertown, Pa.)or medium alone at 37° C. for 30 min. Thereafter, rGal1+/− antibody wasadded to in vitro αCD3 and αCD28 activated T cells. After 16 htreatment, cells were harvested for apoptosis analysis using AnnexinV-FITC and PI (BD Biosciences, San Jose, Calif.) flow cytometry aspreviously described (Juszczynski et al. (2007) Proc Natl Acad Sci USA104:13134-9).

K. Generation of EBV-Transformed Lymphoblastoid Cell Lines (LCLs) andEBV-Specific Cytotoxic T Lymphocytes (CTLs)

After informed consent, 40-60 ml of peripheral blood from healthy donorswas used to generate both EBV-transformed LCLs and EBV-specific CTLs(Straathof et al. (2005) Blood 105:1898-904). In brief, 5×10⁶ peripheralblood mononuclear cells (PBMCs) were incubated with concentrated culturesupernatant from the marmoset B-lymphoblastoid cell line, B95-8, in thepresence of 1 μg/ml cyclosporin A (Sandoz, Vienna, Austria) to establisha LCL. Subsequently, PBMCs (2×10⁶ per well of a 24-well plate) werestimulated with irradiated LCLs (at 4,000 rads) at aneffector-stimulator (E/S) ratio of 40:1. After 10 days, viable cellswere restimulated with irradiated LCLs (at 4:1 E/S ratio). CTLs wereexpanded by weekly stimulations with autologous irradiated LCLs (at 4:1E/S ratio) in the presence of recombinant human interleukin-2 (rhIL-2,Proleukin; Chiron Emeryville, Calif.) at concentration of 40 U/ml. After5 cycles of stimulation, CTLs were tested for EBV specificity andcryopreserved. Specificity was tested using CD107a upregulation as asurrogate marker for CTL degranulation (Betts et al. (2003) J ImmunolMethods 281:65-78).

L. rGal1 Induced Killing of EBV-Specific CTLs

CTLs were thawed in AIM-V media (Invitrogen) containing 10 U/ml of DNAseI (Roche Applied Science, Indianapolis, Ind.) and rested in cultureovernight. The next day, 5×10⁵ CTLs were treated with rGal1 alone orrGal that was pre-incubated with the anti-Gal 1 mAb (8F4F8G7) or theIgG2b isotype control at the indicated concentrations. After 4 hours,the viability of EBV-specific CD8⁺ T cells was measured using 7AAD andAPC-Cγ7-labelled CD8 (BD Biosciences, San Jose, Calif.).

Example 3 Gal1 Expression in EBV-Transformed Lymphoblastoid Cell Linesand Primary Post-Transplant Lymphoproliferative Disorders (PTLDs)

Gal1 transcript abundance was characterized in EBV-transformedlymphoblastoid B-cell lines (LCLs), cell lines from additional B-cellmalignancies including classical Hodgkin lymphoma (cHL), and additionalnormal B cells using publically available gene expression profiles(Basso et al. (2005) Nature Genetics 37:382-90). Gal1 transcripts weresimilarly abundant in EBV-transformed LCLs and cHL cell lines (FIG. 5).For these reasons, Gal1 protein expression was further assessed in aseries of EBV-transformed LCLs using a recently developed anti-Gal1monoclonal antibody, 8F4F8G7 (FIG. 6). All of the examinedEBV-transformed LCLs expressed the ≈14 kd Gal1 protein as did the cHLcell line (FIG. 7A).

A series of primary EBV+PTLDs was next evaluated for Gal1 expression byimmunohistochemical staining; 76% (13/17) of primary EBV+PTLDs wereGal1+ whereas only 4% (3/64) of primary DLBCLs expressed Gal1 (FIG. 7Band Table 3). Similar results were obtained with the Gal1 monoclonalantibody (8F4F8G7, FIG. 7B and Table 3) and the previously describedGal1 antiserum (FIG. 8, (Juszczynski et al. (2007) Proc Natl Acad SciUSA 104:13134-9)).

TABLE 3 Immunohistochemical analysis of Gal1 expression in primary EBV+PTLDs and DLBCLs. Tumors were evaluated by immunohistochemistry with theGal1 mAb, 8F4F8G7, at 1:40,000. Gal1+ Gal1− % Gal1+ EBV+ PTLD 13 4 76DLBCL 3 64 4

Example 4 AP-1 Dependent Gal1 Expression in EBV-Transformed LCLs andPrimary PTLDs

It was previously found that Gal1 expression in classical Hodgkinlymphoma (cHL) was mediated, in part, by an AP-1 dependent Gal1 enhancer(Juszczynski et al. (2007) Proc Natl Acad Sci USA 104:13134-9). BecauseLMP1 and LMP2A both activate the AP-1 pathway and promote the formationof cJun/JunB heterodimers (Kieser et al. (1997) Embo J 16:6478-85; Chenet al. (2002) J Virol 76:9556-61; Song et al. (2004) Cell Signal16:1153-62), the role of the AP-1 dependent Gal1 enhancer inEBV-transformed LCLs was assessed. The abundance and phosphorylation ofthe AP-1 signaling components, cJun and JunB, was first assessed inrepresentative EBV-transformed LCLs (NOR and RIC) by immunoblotting.Total and phosphorylated cJun and JunB were readily detectable in theLCLs and the control cHL cell line (L428) (FIG. 9A). Thereafter, it wasconfirmed that cJun and JunB both bound to the previously described Gal1enhancer (Juszczynski et al. (2007) Proc Natl Acad Sci USA 104:13134-9)in LCLs by ChiP-PCR (FIG. 9B). Densitometric analysis of ChIP-PCRsrevealed that JunB bound Gal1 enhancer regions at higher levels thancJun, highlighting the likely role of JunB as a regulator of Gal1expression. In addition, LCL luciferase activity driven by the Gal1promoter alone or in tandem with the Gal1 enhancer element with anintact or mutated AP-1 binding site was assessed (Juszczynski et al.(2007) Proc Natl Acad Sci USA 104:13134-9). Although the Gal1 promoteralone was active in the NOR LCL cell line, the AP-1-containing enhancerelement increased Gal1-driven luciferase activity (≈3-fold) in an AP-1dependent manner (FIG. 9D).

Having characterized the AP-1-dependent nature of Gal1 expression inEBV-transformed LCLs, AP-1 activity was next evaluated in a cohort ofprimary PTLD tumor specimens. Immunohistochemistry revealed detectableto high-level phospho-cJun expression in all PTLD tumors analyzed(15/15) (FIG. 9E, panels b, d, and f). This was in contrast to primaryDLBCLs, which was previously found to be largely negative forphospho-cJun staining (Rodig et al. (2009) Clin Cancer Res 14:3338-44).Immunohistochemical analysis of JunB revealed uniformly strong nuclearstaining in all PTLD tumors (15/15). (FIG. 9E, panels a, c, and e).Together, these data highlight the role of the AP-1 dependent Gal1enhancer and respective AP-1 components in Gal1 expression inEBV-transformed LCLs and primary PTLDs.

Example 5 Gal1 Promoter Activity in EBV-Transformed LCLs is Driven byLMP-1 and LMP-2a

Given the pivotal role of the EBV latency genes, LMP-1 and LMP-2a, inEBV-induced B-cell transformation (Thorley-Lawson, D A. (2001) NatureReviews Immunology 1:75-82; Kulwichit et al. (1998) Proc Natl Acad SciUSA 95:11963-8; Merchant et al. (2001) Int Rev Immunol 20:805-35), itwas asked whether LMP-1 and LMP-2a modulated Gal1 expression. First,Gal1 transcript abundance in control and LMP-1-transduced normalCD10⁺human germinal center B (GCB) cells was compared using publicallyavailable gene expression profiles (Vockerodt et al. (2008) J Pathol216:83-92) and it was found that Gal1 was ≈2-fold more abundant inLMP-1-transduced GCB cells (FIG. 10). Thereafter, the respective rolesof LMP-1 and LMP-2a in Gal 1 transcriptional activation was evaluated byco-transfecting LMP-1 and/or LMP-2a and a Gal1 promoter-drivenluciferase reporter into 293T cells and evaluating Gal1-drivenluciferase activity. Expression of LMP1 or LMP2a increased Gal1-drivenluciferase activity by ≈4.5- and 2.5-fold, respectively, andco-expression of both LMP proteins was additive (FIG. 11A). Incomplementary studies, siRNA-mediated LMP2a depletion markedly decreasedGal1 expression in an EBV-transformed LCL (NOR) (FIG. 11B). Takentogether, the data directly implicate the EBV proteins, LMP-1 andLMP-2a, in the transcriptional activation of Gal1.

An analysis of the regulatory motifs and modules within the Gal1promoter region was performed and a candidate NFκB binding sitea nd aNFAT/NFY module were identified (FIG. 12); both represent binding sitesfor transcription factors that can be activated by LMP/LMP2a directly(NFκB) or indirectly (NFAT and NFY activation by PI3K/Akt). Havingidentified these putative transcription factor binding sites in the Gal1promoter, inhibitors of NFκB and PI3K/Akt activity were utilized toassess the potential roles of these signaling pathways in Gal 1induction. Overexpression of an IκB super-repressor construct in an LCLcell line (NOR)decreased the abundance of known NFκB target genes, buthad no effect on Gal1 expression. In contrast, treatment of twoEBV-transformed LCLs (NOR and RIC) with a chemical inhibitor of PI3Kactivity (Ly294002) reduced Gal1 expression (FIG. 11D). Taken together,these data indicate that PI3K, but not NFκB, signaling augments Gal1expression in EBV-transformed LCLs.

Example 6 Gal1 Neutralizing mAb Inhibits rGal1-Mediated Killing ofEBV-Specific Cytotoxic T Cells

Given the demonstrated role of Gal1 in tumor immune escape (Rubinsteinet al. (2004) Cancer Cell 5:241-51; Juszczynski et al. (2007) Proc NatlAcad Sci USA 104:13134-9), neutralization of Gal1 activity may representa novel therapeutic strategy for Gal1-expressing tumors. For thisreason, high-titer neutralizing monoclonal antibodies (mAb) directedagainst the Gal1 protein were developed (Example 1). These Gal1 mAbswere first screened for their capacity to inhibit recombinant-Gal1(rGal1)-mediated apoptosis of in vitro activated T cells⁶. The Gal1monoclonal antibody, 8F4F8G7, almost completely inhibited rGal1-inducedapoptosis of normal αCD3/αCD28-activated T cells whereas anisotype-matched control antibody had no effect (FIGS. 13A and 13B).

For these reasons, the effects of 8F4F8G7 on rGal 1-mediated apoptosisof EBV-specific CD8⁺ T cells was assessed. In these assays, rGal1 (1.25,2.5 or 5 μM) was pre-incubated with the neutralizing Gal1 monoclonalantibody (8F4F8G7) or an isotype-matched control (IgG2bλ). Thereafter,EBV-specific, largely CD8⁺, T cells were cultured alone, with rGalalone, or with rGal1 pre-incubated with 8F4F8G7 or the isotype control;following treatment, the percent of viable CD8⁺ (7AAD−) cells wasdetermined (FIG. 14). At all doses, rGal1 alone induced massiveapoptosis of EBV-specific CD8⁺ T cells (FIG. 14, left panel); similarresults were obtained when rGal was preincubated with isotype-matchedcontrol Ig (FIG. 14, right panel). In marked contrast, pre-incubationwith the neutralizing Gal1 mAb (8F4F8G7) almost completely abrogated thecytotoxic effects of rGal1 on EBV-specific CD8⁺ T cells (FIG. 14, middlepanel). Similar results were obtained with EBV-specific CD8⁺ T cellsgenerated from additional independent donors (FIGS. 15A and 15B). Takentogether, these data demonstrate that EBV-specific CD8⁺ T cells areexquisitely sensitive to rGal1-mediated apoptosis and that theneutralizing αGal1 monoclonal antibody, 8F4F8G7, abrogates rGal1-inducedapoptosis of EBV-specific T cells. Therefore, antibody(8F4F8G7)-mediated blockade of secreted Gal1 may represent a novelimmunotherapeutic strategy in EBV-associated PTLD and other Gal1 tumors.

The link between T-cell dysfunction and outgrowth of Epstein-Barr Virus(EBV)-infected B cells is well established (Tran et al. (2008) Blood Rev22:261-81). Herein, it has been demonstrated that the immunomodulatorycarbohydrate-binding lectin, Gal1 is selectively expressed inEBV-transformed LCLs and primary PTLDs and that Gal1 expression isenhanced by EBV-encoded latent membrane proteins and signaling via AP-1and PI3K. Furthermore, a high-titer neutralizing Gal1 mAb has beengenerated that abrogates Gal1-induced apoptosis of EBV-specificcytotoxic T cells. These findings define EBV-associated Gal1 expressionas a novel mechanism of viral immune evasion and highlight the potentialutility of Gal1-neutralizing therapy for PTLD and other Gal1-expressingtumors.

In light of the known capacity of LMP1 and LMP2a to activate AP-1signaling, and our previous description of the AP-1-responsiveness ofGal1 in cHL (Juszczynski et al. (2007) Proc Natl Acad Sci USA104:13134-9; Rodig et al. (2009) Clin Cancer Res 14:3338-44), thebinding of AP-1 signaling components to the Gal1 enhancer inEBV-transformed LCL cell lines aws evaluated. Both cJun and JunB boundthe Gal1 enhancer in EBV-transformed LCLs, to a similar extent as in theL428 HL cell line. Luciferase assays driven by the Gal1 promoter pairedwith either a wild-type or mutated enhancer revealed that AP-1 bindingsites were required for full enhancement of promoter activity.Furthermore, immunohistochemical investigation of AP-1 signalingcomponents in primary PTLD tumors revealed the presence of phospho-cJunand nuclear-localized JunB in all cases, indicating constitutive AP-1activity. These findings therefore indicate that AP-1 signaling may be amechanism of Gal1 induction that is shared by cHL and PTLD.

Luciferase constructs containing only the Gal1 promoter were alsoobserved to be active in an EBV-transformed cell line. As a consequence,the capacity of LMP1/LMP2a signaling to activate the Gal-1 promoter wasevaluated by co-expressing LMP1 and/or LMP2a with the Gal1promoter-driven luciferase construct in an EBV-negative cell line. LMP1and, to a lesser extent, LMP2a increased Gal1 promoter activity and theco-expression of both antigens was additive. In order to characterizethe mechanism by which LMP1/LMP2a activated Gal1 promoter activity, adetailed analysis of regulatory elements within the Gal1 promotersequence was performed and conserved NFκB, NFAT and NFY sites werefound. LMP1 and LMP2a have the potential to induce signaling throughpathways that activate these transcription factors—LMP1 to activate NFκBand both LMP1 and LMP2a to activate NFAT and NFY via PI3K/Akt signaling(Toker et al. (2006) Cancer Res 66:3963-6; Lee et al. (2005) J CellPhysiol 205:270-7). Although molecular or chemical inhibition of NFκBactivity had no effect on Gal1 expression, chemical inhibition of PI3Kmarkedly decreased Gal1 abundance. Therefore, LMP1/LMP2a-associated PI3Ksignaling supports Gal1 expression, likely via subsequent activation ofNFAT and NF-Y. Taken together, these data indicate that Gal1 may beanother gene that is regulated by interactions of NFAT and AP-1 (Macianet al. (2001) Oncogene 20:2476-89).

Evidence presented here and in previous investigations indicate thatGal1 is an important mediator of immune evasion in PTLD, cHL(Juszczynski et al. (2007) Proc Natl Acad Sci USA 104:13134-9), andmelanoma (Rubinstein et al. (2004) Cancer Cell 5:241-51) and that thelectin is also expressed at high level in additional lymphoidmalignancies including anaplastic large cell lymphoma (Rodig et al.(2009) Clin Cancer Res 14:3338-44) and MLL-ALLs (Juszczynski et al.(2010) Clin Cancer Res in press). For these reasons, Gal1 represents anattractive target for directed therapy via mAb-mediated neutralization.Of note, there are ongoing clinical trials of mAb-mediated blockade ofother immune-inhibitory molecules such as PD-1 (Hirano et al. (2005)Cancer Res 65:1089-96) and CTLA-4 (Leach et al. (1996) Science271:1734-6). However, unlike neutralization of CTLA-4, which isassociated with autoimmune-related side-effects in vivo (Sanderson etal. (2005) J Clin Oncol 23:741-50), Gal1-neutralization is expected tobe well tolerated in vivo due to the lack of any observable autoimmunephenotype in Gal1 knock-out mice (Poirier et al. (1993) Development119:1229-36). Furthermore, PTLD is an excellent model system for testingthe utility of a Gal1-neutralizing antibody because LMP-specificcytotoxic T-cells are sensitive to Gal1-induced apoptosis (Smith et al.(2009) J of Virology 83:6192-8).

Therefore, Gal1 specific mAbs were developed and screened for theirability to neutralize rGal1-induced apoptosis of EBV-specific cytotoxicT-cells. Gal1 mAbs that exhibited high affinity and specificity forrecombinant and endogenous Gal1 were first evaluated for their capacityto abrogate rGal1-mediated activated T-cells in vitro. The mosteffective neutralizing Gal1 mAb8F4F8G7, was then assayed against highlyGal1-susceptible, EBV-specific cytotoxic T-cells. Incubation ofEBV-specific donor cytotoxic T-cells with 8F4F8G7 dramatically reducedrGal1-mediated apoptosis compared to the isotype control antibody,highlighting the potential utility of this mAb in Gal1-neutralizingtherapy.

In summary, it has been shown that EBV-transformed LCLs and primaryPTLDs exhibit strong expression of Gal1 that is promoted by the LMP1 andLMP2a viral antigens through PI3K/Akt and AP-1 signaling. In addition, aGal1-neutralizing mAb that protects against rGal1-induced apoptosis ofEBV-specific cytotoxic T-cells was generated. Taken together, theseresults demonstrate a novel mechanism for EBV-induced immune evasion inPTLD and indicate an associated targeted therapeutic strategy for thisdisease and other Gal1-expressing malignancies.

Example 7 Materials and Methods for Examples 8-12

A. Mice

Lgals1^(−/−) mice (C57BL/6) were provided by F. Poirier. SwissN:NIH(S)nu (nude) mice were obtained from the University of La Plata andB6/Rag^(/) mice were from Jackson Lab. Mice were bred at the animalfacilities of the Institute of Biology and Experimental Medicineaccording to NIH guidelines. Protocols were approved by the respectiveInstitutional Review Boards.

B. Cells

KS-1 mm is a spontaneously immortalized cell line obtained from a KSbiopsy as described (Albini et al. (2001), Cancer Res 61, 8171-8178).All other cell lines were obtained from the ATCC. Primary HUVEC weremaintained in M-199 medium supplemented with 20% FCS, EGF (10 ng/ml),bFGF (10 ng/ml), VEGF (20 ng/ml) (all from R&D) and used between passage2 and 5. Gal1-specific shRNA was designed and cloned into thepSIREN-RetroQ vector as described in Juszczynski et al. (2007) Proc NatlAcad Sci USA 104, 13134-13139.

C. Glycophenotypic Analysis, Galectin Binding and Segregation Assays

For glycophenotyping, ECs were incubated with biotinylated L-PHA, LEL,SNA, MAL II, PNA and HPA (all from Vector). Recombinant Gal1 waspurified as described in Ilarregui et al. (2009) Nat Immunol 10,981-991. For binding assays, ECs were incubated for 1 h at 4° C. withdyLight 488-labeled galectins in the absence or presence of lactose,sucrose, anti-Gal1 or isotype control mAb or following transfection withGnTS, GCNT1 or scrambled siRNA. Cells were analyzed on a FACSAria (BDBiosciences). For segregation, ECs were treated with Gal1 for 1 h, fixedand incubated for 1 h with anti-human VEGFR2 antibody (55B11; CellSignaling) as described in Ilarregui et al. (2009) Nat Immunol 10,981-991. Cells were analyzed on a Nikon laser confocal microscope(Eclipse E800).

D. Angiogenesis Assays

HUVEC transfected or not with specific siRNA or pre-incubated withsignaling pathway inhibitors were exposed to VEGF or Gal1 with orwithout lactose, sucrose, 8F4F8G7 mAb or control isotype (IgG1κ) orspecific antibodies for VEGFR1 (AP-MAB0702; Abcam), VEGFR2 (AF357; R&D),VEGFR3 (AB89501; Abcam) or VEGF (MAB293; R&D). Cells were processed forproliferation, migration, invasion and tube formation assays.Tumor-associated blood vessels were identified by flow cytometry usingAlexa Fluor 647-conjugated anti-CD34 antibody (RAM34; eBioscience).

E. Phospho-RTK Signaling Array, Co-Immunoprecipitation andImmunoblotting

Cells were lysed and analyzed by the human PathScan® RTK SignalingAntibody Array (Cell Signaling) following manufacturer's directions. Forco-immunoprecipitation, 500 μg cell lysates were incubated with 2 μg ofanti-VEGFR2 (55B11; Cell Signaling) or anti-NRP-1 (C-19; Santa CruzBiotechnol) antibodies. The immunocomplexes were captured with protein GPLUS-Agarose (Santa Cruz Biotechnol) and processed for immunoblotanalysis as described in Ilarregui et al. (2009) Nat Immunol 10,981-991. Equal amounts of protein were resolved by SDS-PAGE, blottedonto nitrocellulose membranes (GE Healthcare) and probed with anti-IκB-α(C21), anti-Erk1/2 (C14), anti-phospho-Erk1/2 (E4) or anti-actin (I-19)(all from Santa Cruz Biotechnol) or anti-Akt (9272), anti-phospho-Akt(9271S), anti-VEGFR2 (55B11), anti-phospho-VEGFR2 (19A10) (all from CellSignaling) or anti-HIF-1α (mgc3; ABR BioReagent) antibodies or a rabbitanti-Gal1 IgG (1.5 μg/ml) obtained as described in Tlarregui et al.(2009) Nat Inmmunol 10, 981-991.

F. In Vivo Tumor Models

Wild-type KS cells or shRNA clones (5×10⁶ cells) were injectedsubcutaneously into 6- to 8-week old nude mice. Wild-type B16 cells orshRNA clones (2×10⁵ cells) were injected into 6- to 8-week old B6 orB6/Rag^(−/−) mice and tumor development was monitored as described inRubinstein et al. (2004) Cancer Cell 5, 241-251. Treatments with 8F4F8G7mAb or control isotype (2.5, 7.5 or 15 mg/kg; i.p. injections everythree days) were initiated when tumors reached 100 mm³. Mice weresacrificed when tumors reached a volume greater than 2 cm³. At 2 weeksafter tumor challenge, lymph node cells (5×10⁵ cells/well) wererestimulated for 72 h with 1×10⁴ irradiated (4,000 rads) B16 cells andwere analyzed for proliferation and cytokine production. For adoptivetransfer, splenic T cells (5×10⁶) from tumor-bearing mice were labeledwith CFSE (Molecular Probes) and injected through the tail vein intotumor-bearing recipient mice treated with 8F4F8G7 or control mAb. Inrelated studies, fluorescent beads (3 μm; BD Biosciences), rather thansplenic T cells, were injected into tumor-bearing animals treated with8F4F8G7 or control antibody. CFSE cells or PerCP-labeled beads wereanalyzed after 24 h or 15 min respectively by flow cytometry in tumorparenchyma and spleen.

G. Immunohistochemistry and Confocal Microscopy

For immunostaining, mice were anesthetized and cardiac-perfused with PBSand 4% paraformaldehyde and tissues were embedded in OCT. To visualizethe vasculature, mice were intravenously injected with FITC-conjugatedGriffonia simplicifolia Lectin-1 (GLS-1_(B4); Vector) prior to heartperfusion and fixation. Pericyte maturation was assessed usingantibodies specific for αSMA (1A4; Dako), desmin (D33; Dako), PDGFRβ(APBS; Biolegend) and Rgs5 (HPA001821; Prestige Sigma). The fraction ofpericyte coverage was calculated as the ratio of αSMA area to theFITC-GLS-1_(B4) or CD31 stained area using a specific anti-CD31 antibody(Mec13.3; BD Biosciences). For immunoperoxidase staining,paraffin-embedded human tumor sections were incubated with anti-CD31(JC/70A; Dako) and anti-Gal1 antibodies as described in Juszczynski etal. (2007) Proc Natl Acad Sci USA 104, 13134-13139 using the VectastainElite ABC kit (Vector). Studies with patient biopsies were subjected toInstitutional Review Boards approval (CEMIC and IBYME). Hypoxia wasdetected after injection of pimonidazole hydrochloride for 30 minfollowing immunostaining with Hypoxyprobe-1 plus kit (Natural PharmaciaInternational).

H. Generation of Anti-Galectin-1 mAb

The neutralizing Gal-1 mAb was generated and characterized as describedherein.

I. Statistical Analysis

Prism software (GraphPad) was used for statistical analysis. Two groupswere compared with the Student's-t test for unpaired data. Two-way ANOVAand Dunnett's or Tukey post-tests were used for multiple comparisons.Nonparametric analysis was performed using Mann-Whitney Utest. P valuesof 0.05 or less were considered significant.

J. Reagents

DyLight 488-conjugated Gal1 (488-Gal1) was obtained using DyLightlabeling kit (Thermo Scientific). Inhibitors of Jak2-STAT3 (AG490; 5μM), Jnk-SAP (SP600125; 20 p38 (SB202190; 10 μM), HIF-1α (3 μM) andβ-glycosylation (benzyl-α-GalNAc; 2 mM) were from Calbiochem. Inhibitorsof Erk1/2 (U0126; 5 μM), PI(3)K-Akt (Ly294002; 2 μM) and NF-κB (BAY11-7082; 1 ROS(N-acetyl-cysteine; NAC), N-glycosylation (swainsonine; 3lactose or sucrose (30 mM) were from Sigma. PNGase F (25 U/μg protein)was from New England Biolabs. Recombinant cytokines including IL-10 (50ng/ml), IL-17 (5 ng/ml), TGF-β₁ (3 ng/ml), IFN-γ (50 ng/ml), VEGF (20ng/ml), bFGF (10 ng/ml) were from R&D. TNF (20 ng/ml) was from Sigma.Biotinylated lectins, including L-PHA (2 μg/ml), LEL (1 SNA (5 μg/ml),MAL II (10 μg/ml), PNA (10 μg/ml) and HPA (10 μg/ml) were purchased fromVector Labs and incubated in buffer containing 150 mM NaCl, 10 mM HEPESand 1% BSA (Sigma). ON-TARGETplus SMART siRNA pools against GnT5, GCNT1,VEGFR2, NRP-1, VEGF, HIF-1α and scrambled were obtained from Dharmacon.Transfections were performed by Lipofectamine-RNAiMAX (Invitrogen)following manufacturer's directions. Recombinant Gal3 and Gal8 werepurified as described in Acosta-Rodriguez et al. (2004) J Immunol 172,493-502 and Cardenas Delgado et al. (2010) FASEB J [Epub ahead ofprint].

K. Cells and Knockdown Clones

The A375 and LNCaP cell lines were cultured in RPMI-1640 GlutaMaxcomplete medium supplemented with 10% FCS and the B16-F0 and 4T1 cellswere cultured in DMEM supplemented with 5% FCS (all from Gibco).Retroviral shRNA delivery was performed using RetroPack PT-67 packagingcell line (BD Biosciences) according to manufacturer's instructions.After infection, cells were subjected to puromycin selection (5 μg/ml)and clones were obtained by limited dilution. For the antisensestrategy, subconfluent KS cells were transfected with the pcDNA6/Gal1antisense vector created as described in Rubinstein et al. (2004) CancerCell 5, 241-251 and cloned by limited dilution. The in vitro growth ofrelevant clones was measured by the MTS assay (Promega).

L. Angiogenesis Assays

The formation of capillary-like tubular structures was assessed inMatrigel-coated plates essentially as described in Albini et al. (2001),Cancer Res 61, 8171-8178. In brief, HUVEC (3×10⁴ cells/ml) transfectedor not with specific siRNA or pre-incubated with signaling pathwayinhibitors were seeded on Matrigel with our without Gal1 (0.1 to 3 μM)or VEGF (20 ng/ml) with or without lactose, sucrose, 8F4F8G7 mAb (0.5μM) or isotype control (IgG1κ) or blocking antibodies specific forVEGFR1 (5 μg/ml), VEGFR2 (2 μg/ml), VEGFR3 (10 μg/ml) or VEGF (10μg/ml). Cells were incubated at 37° C. for periods ranging from 0 to 24h and were visualized by phase-contrast microscopy. In another set ofexperiments, conditioned medium from hypoxic or normoxic KS cellsinfected or not with a retroviral vector containing Gal1 shRNA wasassessed on HUVEC transfected or not with GnT5, GCNT1 or scr siRNA (100nM). Capillary-like tubular structures were scored by counting thenumber of tubules (closed areas) per well in a phase-contrast microscope(Nikon E-100). For migration assays, HUVEC (4×10⁴/well) transfected ornot with specific siRNA were resuspended in M199 medium supplementedwith 1% FCS. Cells were placed into the top chamber of the insert whilethe bottom well was filled with Gal1 or VEGF in the absence or presenceof lactose, sucrose, 8F4F8G7 mAb or isotype control. After 24 hours,inserts were stained with crystal violet (Sigma) and analyzed in aninverted microscope. For each filter, 4 images were collected and cellswere counted with the ImageJ software v1.34 (NIH).

For proliferation assays, HUVEC that were transfected or not withspecific siRNA and cultured in complete M199 medium were trypsinized,harvested and seeded in 96-well microtiter plates (1×10³ cells/well).Cells were pre-incubated for 1 hour at 37° C. with lactose, sucrose orsignaling pathway inhibitors and then exposed to Gal1. After 24 hours,cells were incubated for an additional 24 hours in the presence of 0.8μCi [³H]-thymidine (NEN Dupont). Cells were then harvested andradioactivity was measured in a 1414 Liquid Scintillation Counter(Perkin Elmer).

Invasion assays were performed using the BioCoat Angiogenesis system (BDBiosciences) following manufacturer's recommendations. For assessment ofin vivo angiogenesis, growth factor-reduced Matrigel (BD Biosciences)was mixed with Gal1 (0.1 or 3 μM) with or without lactose (30 mM). Inanother set of experiments, serum-free CM from KS cells infected or notwith a retroviral vector expressing Gal1 shRNA and cultured underhypoxic or normoxic conditions, were added to unpolymerized Matrigel andinjected subcutaneously into the flanks of either wild-type orLgals1^(−/−) (B6) mice or nude mice using a cold syringe. Matrigelembedded with buffer alone was used as negative control and a cocktailcontaining VEGF (50 ng/ml), heparin (50 U/ml) and TNF (2 ng/ml) was usedas positive control. After 6 days, Matrigel plugs were collected bysurgery, photographed and weighed. Samples were minced and diluted inwater to measure hemoglobin content using the Drabkin reagent kit(Sigma). Each sample was normalized to 100 mg of recovered gel andconfronted with a standard curve of mouse blood hemoglobin.

M. Real-Time Quantitative RT-PCR

SYBR® Green PCR Master Mix was used with an ABI PRISM® 7500 SequenceDetection Software (all from Applied Biosystem). Primers used were:human Gal1 forward: 5′-TGAACCTGGGTAAAGACA-3′ (SEQ ID NO: 20); reverse:5′-TTGGCCTGGTCGAAGGTGAT-3′ (SEQ ID NO: 21); human RN18S1 forward:5′-CGGCCGGGGGCATTCGTATT-3′ (SEQ ID NO: 22); reverse:5′-TCGCTCTGGTCCGTCTTGCG-3′ (SEQ ID NO: 23); human GCNT1 forward:5′-CCTCCTGAGACTCCGGGGTCAGA-3′ (SEQ ID NO: 24); reverse:5′-CTAGGCGGTCCGTGCCCTAGC-3′ (SEQ ID NO: 25); human GnT5 forward:5′-TGCCCCTGCCGGGACTTCAT-3′ (SEQ ID NO: 26); reverse:5′-CAGCAGCATGGTGCAGGGCT-3′ (SEQ ID NO: 27).

N. Analysis of LGALS1 Promoter Constructs with Luciferase Assays

Cells transfected or not with HIF-1α siRNA (100 nM) or IκB-α-SR (500 ng)were grown to 60-80% confluence on 24-well plates and co-transfectedwith 500 ng pGL3-Gal-Luc vector containing the LGALS1 promoter region(−473 to +67) ligated into the pGL3 promoterless reporter vector(Promega) and 20 ng of the control reporter plasmid pRL-TK (Promega)using FUGENE® HD (non-liposomal lipid blend formulated in 80% ethanol)transfection reagent (Roche Applied Science) according to manufacturer'srecommended protocol. After 24 hours, culture medium was replaced forM199 1% FCS and cells were incubated under normoxic or hypoxicconditions in the absence or presence of NF-κB or HIF-1α inhibitors.After 18 hours, cells were lysed and luciferase activity was determinedby chemiluminiscence using the dual luciferase assay kit (Promega) in a20/20^(n) luminometer (Turner Biosystem).

O. Analysis of Regulatory Elements in the LGALS1 Locus

Computational analysis of the LGALS1 locus (2400 bp upstream to 2500 bpdownstream to the start site) was performed with the publicly availableversion of MatInspector software (available on the world wide web at theGenomatix website and multiple κB binding sites were identified (FIG.18P).

P. Induction of Hypoxia

Tumor cell lines or HUVEC were cultured in 24-well plates, placed in amodular incubator chamber (Billups-Rothenberg) and flushed at 2 psi for10 min with a mixture of 1% O₂, 5% CO₂ and 94% N₂. The chamber wassealed and placed in a 37° C. incubator for 18 hours. Controls ofnormoxia were placed in the same incubator at 5% O₂. Chemical inductionof HIF-1α (a condition often termed ‘pseudo-hypoxia’) was inducedfollowing treatment with CoCl₂ (Sigma).

Q. Intracellular Staining and FACS Analysis For intracellular cytokinestaining, TLDN or tumor-infiltrating lymphocytes were made permeablewith Perm2 solution (BD Biosciences) and were labeled withfluorescent-labeled monoclonal anti-IFN-γ (XMG1.2; BD Biosciences),anti-IL-17 (TC11-18H10; BD Biosciences), anti-IL-10 (JES5-16E3;eBioscience), anti-CD4 (GK1.5; BD Biosciences), anti-CD8 (H35-17.2;eBioscience) antibodies. T_(reg) cells were determined by using themouse T_(reg) staining kit (FJK-16s, eBioscience). Cells were analyzedon a FACSAria (BD Biosciences) using a FlowJO software.R. ELISA

Mouse IFN-γ and IL-10 ELISA sets were from BD Biosciences and mouseIL-17 kit was from R&D. Human soluble VEGF was assessed by ELISA(DY293B; R&D). Soluble Gal1 was determined using an in-house ELISA.Briefly, high binding 96-well microplates (Corning Costar) were coatedwith capture antibody (2 μg/ml purified rabbit anti-Gal1 polyclonal IgG)in 0.1 M sodium carbonate pH 9.5. After incubation for 18 hours at 4°C., wells were rinsed three times with wash buffer (0.05% Tween-20 inPBS) and incubated for 1 h at RT with blocking solution (2% BSA in PBS).Samples and standards (100 μl) were diluted in 1% BSA and incubated for18 hours at 4° C. Plates were then washed and incubated with 100 ng/mlbiotinylated detection antibody (purified rabbit anti-Gal1 polyclonalIgG) for 1 hour at RT. Plates were rinsed three times before addinghorseradish peroxidase-labeled streptavidin (0.33 μg/ml; Sigma) for 30min at RT. After washing, 100 μl of TMB solution (0.1 mg/mltetramethylbenzidine and 0.06% H₂O₂ in citrate-phosphate buffer pH 5)was added to the plates. The reaction was stopped by adding 4NH₂SO₄.Optical densities were determined at 450 nm in a Multiskan MS microplatereader (Thermo Electron Corporation). A standard curve ranging from 2.5to 160 ng/ml recombinant Gal1 was run in parallel.

S. Confocal Microscopy and Immunohistochemistry

For confocal microscopy the following primary antibodies were used:mouse anti-αSMA (1A4; Dako; 1:100), mouse anti-desmin (D33; Dako;1:100), rat anti-PDGFRβ (APB5; Biolegend; 1:50), rabbit anti-Rgs5(Prestige Sigma; 1:50), rat anti-CD31 (Mec13.3; BD Biosciences; 1:100),rabbit anti-Gal1 IgG (1:100) generated as described in Ilarregui et al.(2009) Nat Immunol 10, 981-991, rabbit anti-VEGFR2 (55B11; CellSignaling; 1:200), mouse anti-CD8 (H35-17.2; eBioscience; 1:50), ratanti-LANA (Advanced Biotechnol; 1:1000). Secondary antibodies used were:anti-mouse IgG-FITC (BD Biosciences; 1:200), anti-mouse IgG-Cγ3 (Vector;1:500), anti-rat IgG-FITC (Vector; 1:500), anti-rat IgG-Texas Red(Vector: 1:500) and anti-rabbit IgG-Alexa Fluor-555 (Cell Signaling:1:1000).

T. Microarrays of KS and Data Analysis

The Human Genome Array Hg-U133A (Affymetrix) (Wang et al. (2004) NatGenet 36, 687-693) and the Mouse Genome 430 2.0 Array (Affymetrix) wereused to examine gene expression levels of KS biopsies and mECK36 tumorsas described in Mutlu et al. (2007) Cancer Cell 11, 245-258. Raw dataintensity profiles were analyzed using the GeneSpring 7 (Agilent) toperform microarray normalization and statistical analysis.

Example 8 Regulated Glycosylation Modulates Vascular Biology by Allowingthe Formation of Lectin-Glycan Lattices

To study whether galectin-saccharide lattices contribute to formation oftumor vascular networks, the ‘glycosylation signature’ of humanendothelial cells (ECs) was first examined both under resting conditionsand when ECs are exposed to proliferative, tolerogenic or inflammatorystimuli. For this purpose, a panel of lectins was used which selectivelyrecognize glycan structures, including those that are relevant for Gal1binding and signaling (FIG. 16K). Gal1 recognizes multiplegalactose-β1-4-N-acetylglucosamine (LacNAc) units, which may bepresented on the branches of N- or O-linked glycans (Hirabayashi et al.(2002) Biochim Biophys Acta 1572, 232-254). Thus, regulated expressionof glycosyltransferases during vascular remodeling, that serve to createpoly-LacNAc ligands, may determine susceptibility to Gal1. This includesthe N-acetylglucosaminyltransferase 5 (GnT5), an enzyme that generatesβ1,6-N-acetylglucosamine-branched complex N-glycans (Dennis et al.(2009) Cell 139, 1229-1241). Under resting conditions, primary humanvein umbilical ECs (HUVEC) showed considerable expression ofL-phytohemagglutinin (L-PHA)-reactive GnT5-modified N-glycans (FIG. 16Awhich substantially increased following exposure to IL-10 or TGF-β₁,both cytokines capable of imprinting anti-inflammatory or tolerogenicsignatures (FIG. 16B). A similar tendency was observed followingstimulation with a strong proliferative stimulus such as basicfibroblast growth factor (bFGF) (FIG. 16B). In contrast, ECs stimulatedwith pro-inflammatory (tumor necrosis factor; TNF), T_(H)1-type (IFN-γ)or T_(H)17-type (IL-17) cytokines showed a significant reduction ofL-PHA-reactive glyco-epitopes (FIG. 16B). Staining with the Lycopersiconesculentum lectin (LEL), which recognizes poly-LacNAc ligands, revealeda substantial increase in reactive glyco-epitopes following exposure totolerogenic and proliferative stimuli (FIGS. 16A and 16B). As α2-6sialyltransferase (ST6Gal1) may modify LacNAc ligands and block Gal1signaling (Toscano et al. (2007) Nat Immunol 8, 825-834), binding of theSambucus nigra agglutinin (SNA), a lectin that recognizes α2-6-linkedsialic acid (SA) sequences, was examined. ECs stimulated with bFGF or acombination of IL-10 and TGF-β₁ responded with diminished display ofSNA-reactive glyco-epitopes, as compared to resting, TNF-, IL-17- orIFN-γ-treated ECs (FIGS. 16A and 16B), indicating that pro-inflammatoryor anti-inflammatory signals may either mask or unmask poly-LacNAcsequences. In contrast, human ECs showed similar binding profiles forthe Maackia amurensis agglutinin (MAL II), which recognizes α2-3 SAlinkages, regardless of the stimuli used (FIGS. 16A and 16B); theseresults indicate that changes in glycosylation are specific and do notrepresent global loss of SA from cell surface glycoproteins.

Binding of Gal1 may also be regulated by glycosyltransferases, whichcompete for acceptor substrates and thus limit carbohydrate ligandsynthesis. The α2-3 sialyltransferase I (ST3Gal1) competes with thecore-2 β1-6-N-acetylglucosaminyltransferases (GCNTs) for core-1 O-glycanstructures and may inhibit the addition of O-linked poly-LacNAc ligands(FIG. 16K). To assess the influence of this pathway, EC surfaceglyco-receptors were probed for the absence of sialylated core-1O-glycans using the lectin peanut agglutinin (PNA), which binds toasialo-galactose-β1-3-N-acetylgalactosamine core-1 O-glycans. Exposureof human ECs to bFGF or IL-10 resulted in a modest but significantincrease in PNA-reactive asialo core-1 O-glycans, compared to cellsexposed to pro-inflammatory, T_(H)1 or T_(H)17 stimuli (FIGS. 16A and16B). Finally, no significant binding of Helix pomatia (HPA), a lectinthat recognizes terminal α-N-acetyl-galactosamine residues was observed(FIG. 16A). In most cases the combination of tolerogenic oranti-inflammatory stimuli had additive effects (FIG. 16B). Similarresults were observed using the murine EC line, EOMA. Collectively,these results indicate that proliferative and tolerogenic stimuli,commonly found in tumor microenvironments, favor a ‘Gal1 permissive’glycophenotype on ECs, while pro-inflammatory signals tend to interruptexposure of these glyco-epitopes. These results emphasize the dynamicsof the EC surface ‘glycome’, which may contribute to vascular biologythrough either masking or unmasking specific glyco-epitopes forendogenous lectins.

To determine whether the regulated glycan repertoire facilitates theformation of galectin-glycan lattices, binding of fluorescently-labeledGal1 to ECs was analyzed under different experimental conditions. Gal1bound to ECs in a dose- and carbohydrate-dependent fashion; the specificdisaccharide lactose, but not sucrose, prevented these interactions(FIG. 16C). To dissect the contribution of N- and O-glycans to Gal1effects, binding assays were performed in the absence or presence ofglycosylation pathway inhibitors. Binding of Gal1 to ECs was almostcompletely abrogated by swainsonine, an early inhibitor of N-glycanbiosynthesis, whereas benzyl-α-GalNAc, a metabolic competitor ofO-glycan elongation, was only partially inhibitory (FIG. 16C). Moreover,interruption of complex-type N-glycan branching through shortinterfering RNA (siRNA)-mediated silencing of GnT5 almost completelyeliminated Gal1 binding to the surface of ECs, whereas inhibition ofcore 2 O-glycan elongation through siRNA-mediated silencing of GCNT1 hadno effect (FIGS. 16D and 16L-16N), clearly demonstrating the glycanspecificity of this effect. Consistent with changes in glycosylation,binding of Gal1 was much higher in ECs exposed to proliferative ortolerogenic stimuli (either alone or in combination) compared to cellssensing inflammatory, T_(II)1 or T_(II)17 signals (FIG. 16E). Thus,highly branched cell surface N-glycans may influence vascular biologythrough the formation of discrete Gal1-glycan lattices, which arepreferentially established under tolerogenic or proliferative settings.

To examine the functional relevance of these interactions, whether Gal1controls vascular biology through a glycosylation-dependent mechanismwas determined. Signaling through Gal1-glycan lattices elicited thetypical cellular processes associated with angiogenic sprouting,including EC proliferation, migration and invasion and enabled theformation of three-dimensional tubular networks at levels similar tothose attained by VEGF (FIGS. 16F-16H and 16O-16S). These effects werecompletely prevented by addition of the specific disaccharide lactose orby siRNA-mediated GnT5 silencing, while introduction of GCNT1 siRNA hadno effect (FIGS. 16F-16H), indicating a critical role for LacNAcresidues and complex N-glycan branching in angiogenic sprouting mediatedby Gal1. However, the pro-angiogenic effects of VEGF were preservedregardless of the absence or presence of N- or O-glycan branching (FIG.16I). In vivo, injection of Matrigel sponges containing recombinant Gal1rapidly became vascularized in a manner that was dose-dependent andspecifically inhibited by lactose (FIG. 16J). Thus, unlike VEGF, Gal1endows ECs with pro-angiogenic potential through mechanisms involvingregulated glycosylation of putative signaling receptors.

Example 9 Galectin-1 Co-Opts VEGFR2 Signaling Pathways Through theFormation of Lectin-Glycan Lattices on Highly Branched Xomplex N-Glycans

To elaborate further on the mechanisms associated with thepro-angiogenic functions of Gal1-glycan lattices and to identifyputative glyco-receptors mediating these effects, changes in thephosphorylation status of a spectrum of growth factor receptor tyrosinekinases (RTKs) and signaling nodes were screened using a phospho-RTKsignaling array. The only RTK that became phosphorylated followingtreatment of human ECs with Gal1 was VEGFR2 (FIGS. 17A and 17K). Thisphosphorylation pattern was detected as early as 15 min (FIG. 17A) andwas sustained even after 60 min of exposure to this lectin. In addition,Gal1 exposure increased the phosphorylation of Akt (Thr308), Akt(Ser473) and the mitogen-activated protein kinase Erk1/2, recapitulatingthe phosphorylation pattern elicited by VEGF (FIG. 17A). Dose-dependentphosphorylation of VEGFR2, Akt and Erk1/2 was further validated byimmunoblot analysis (FIG. 17B). Accordingly, pharmacological inhibitionof PI(3)K/Akt or Erk1/2 completely suppressed Gal1-induced ECproliferation, migration and tubulogenesis, while inhibition of Jnk,p38, STAT3 or NF-κB had no effect (FIGS. 17C-17E). Furthermore,siRNA-mediated silencing of VEGFR2 completely prevented Akt and ERK1/2phosphorylation induced by either Gal1 or VEGF (FIGS. 17F and 17L). Asbranching of complex N-glycans attached to growth factor receptors mayfine-tune the threshold for growth factor signaling (Lau et al. (2007)Cell 129, 123-134; Song et al. (2010) Cancer Res 70, 3361-3371),analyses were conduected to determine whether fluctuations inGnT5-modified glycans can directly modulate sensitivity of VEGFR2 to itscognate ligand VEGF. Silencing of GnT5-mediated N-glycan branchingselectively eliminated Gal1, but not VEGF signaling (FIG. 17F). Incontrast, blockade of core-2 O-glycan elongation via GCNT1 knock-downhad no substantial effect. These results indicate that Gal1 and VEGFR2selectively associate to generate multivalent signaling clusterscharacterized by the presence of highly branched complex N-glycans.

To determine whether Gal1 establishes direct interactions with VEGFR2through N-glycosylation-dependent mechanisms, co-immunoprecipitationexperiments with lysates of human ECs treated with Gal1 were performedin the absence or presence of PNGase F, an endoglycosidase that releasesN-linked oligosaccharides, or following transfection with GnT5 siRNA tointerrupt complex N-glycan branching Gal1 associated specifically withVEGFR2 through interactions that depended on early or late stages ofN-glycan elongation (FIG. 17G). Supporting these findings, exposure toGal1 resulted in segregation of VEGFR2 to membrane patches, indicatingrearrangement of signaling clusters on the surface of human ECs.Segregation was eliminated following siRNA-mediated GnT5 silencing (FIG.17H). Hence, rather than altering VEGF signaling, Gal1 directly co-optsthe VEGFR2 signaling pathway through binding to LacNAc-enriched complexN-glycan structures.

Given the contribution of various glyco-receptors to angiogenic switch(Lemmon et al. (2010) Cell 141, 1117-1134), their involvement inGal1-mediated effects was also examined. Inhibition of VEGFR2 signalingthrough siRNA-mediated silencing or through antibody-mediated blockadeabrogated Gal1-induced EC migration and tube formation, whereas blockadeof VEGFR1 or VEGFR3 had no effect (FIGS. 17I, 17J, 17P, and 17Q),indicating that only selected glyco-receptors are amenable to theformation of signaling clusters mediated by lectin-glycan lattices.Moreover, siRNA-mediated silencing of NRP-1, a transmembraneglycoprotein responsible for amplifying VEGFR2 signaling, did notsignificantly affect Gal1-induced tube formation, in spite of itsability to interact with this lectin (FIGS. 17I, 17M, and 17N). Whileinhibition of VEGFR2 signaling abrogated EC migration induced by Gal1 orVEGF, NRP-1 silencing suppressed only VEGF effects (FIG. 17P).

Because of the active search for VEGF-independent angiogenic pathwaysand the autocrine effects of VEGF signaling (Lee et al. (2007) Cell 130,691-703), it was next investigated whether Gal1-VEGFR2 signalingproceeded in the absence of VEGF. Consistent with lack of effects ofGal1 on VEGF secretion (FIG. 17R), inhibition of VEGF signaling throughsiRNA-mediated silencing or antibody-mediated blockade did not preventGal1-induced EC migration and tube formation (FIGS. 17I, 17J, and17O-17Q). Collectively, the results indicate that signaling complexesestablished between endogenous lectins and specific glycan structures onselected growth factor receptors mimic ‘canonical’ ligands to preservecritical cellular processes, including angiogenesis.

Example 10 Galectin-1-Glycan Lattices Link Tumor Hypoxia toVEGFR2-Mediated Angiogenesis

Despite considerable progress in elucidating the signaling pathways thatcontrol hypoxia and angiogenesis, the molecular mechanisms couplingthese processes are still poorly understood. To investigate whetherGal1-glycan lattices link tumor hypoxia to sprouting angiogenesis,whether exposure to hypoxic microenvironments can influence the‘glycosylation signature’ of ECs was determined. As revealed byglycophenotypic analysis, hypoxia (1% O₂) induced a substantial increasein β1-6-branched complex-type N-oligosaccharides (L-PHA reactivity) andpoly-LacNAc structures (LEL reactivity) concomitant with a considerablereduction in α2-6-linked SA (SNA binding) and slight changes inasialo-core-1 O-glycans (PNA binding), as compared to ECs cultured undernormoxic conditions (FIG. 18A). In contrast, no significant differenceswere observed in α2-3 sialylation (MAL II reactivity) between ECssubjected to hypoxia or normoxia (FIG. 18A). These data indicate anextensive and selective remodeling of the EC surface ‘glycome’ inresponse to hypoxia, similar to that found in response to tolerogenic orproliferative stimuli, which results in increased availability of cellsurface glycans essential for Gal1 signaling. Accordingly, preferentialbinding of this lectin to ECs exposed to hypoxia was found, as comparedto those incubated under normoxic conditions (FIG. 18B).

To further delineate the functional role of Gal1-glycan lattices inhypoxic microenvironments, the regulated expression of tumor-derivedGal1 under hypoxic or normoxic conditions in immortalized Kaposi'ssarcoma (KS) cells, which typically develop tumors characterized by adense and poorly organized capillary network recruited from the host(Albini et al. (2001), Cancer Res 61, 8171-8178), was analyzed. Hypoxiainduced considerable up-regulation of Gal1 in KS cells, as shown by the2-fold induction of LGALS1 promoter activity, 4-fold induction of Gal1mRNA and 2.5-fold induction of protein expression and secretion, ascompared to KS cells grown in normoxic conditions (FIGS. 18C-18F).Hypoxia-induced Gal1 expression was also evident in human and murinemelanoma (A375 and B16-F0), mouse breast carcinoma (4T1) and humanprostate carcinoma (LNCaP) cell lines (FIG. 18M), indicating broadregulation of endogenous Gal1 at the transcriptional level in tumors ofmesenchymal or epithelial origin. This effect was independent of themaster transcription factor HIF-1α as hypoxia still inducedup-regulation of Gal1 in either KS cells transfected with HIF-1α siRNA(FIGS. 18C-18F) or in KS cells incubated with a specific HIF-1αinhibitor (FIG. 18N).

Consistent with these results, chemical activation of HIF-1α (withCoCl₂) had no effect on Gal1 expression (FIG. 18O). As bothHIF-dependent and HIF-independent oxygen-sensing mechanisms have beenlinked to NF-κB-regulated gene transcription (Rius et al. (2008) Nature453, 807-811), it was next asked whether hypoxia controls Gal1expression through NF-κB-regulated pathways. Blockade of NF-κBtranscriptional activity by expression of a super-repressor form ofIκB-α (IκB-α-SR) or pharmacological inhibition using BAY-117802prevented IκB-α degradation and completely eliminated hypoxia-drivenGal1 expression and secretion without altering the levels of HIF-1α(FIGS. 18C-18F and 18N). Supporting these findings, analysis of theregulatory sequences of human LGALS1 gene revealed several putativeNF-κB consensus sequences (FIG. 18P), including a specific site locatedat the promoter sequence -341 bp upstream of the start site, which wasfunctionally active in transcriptional assays (FIG. 18C). As NF-κBactivation may result from oxidative stress of hypoxic cells due to thegeneration of reactive oxygen species (ROS; Mizukami et al, 2005),whether hypoxia induces NF-κB activation and subsequent up-regulation ofGal1 through increased production of ROS was determined. Scavenging ofROS using N-acetyl-cysteine (NAC) strongly inhibited induction of Gal1expression and secretion and prevented IκB-α degradation in KS cellscultured under hypoxic conditions (FIGS. 18G and 18Q). Moreover,exogenous administration of H₂O₂ stimulated the secretion of Gal1 in adose- and NF-κB-dependent fashion (FIGS. 18H and 18R). These dataindicate that ROS-dependent activation of NF-κB, but not HIF-1α,controls the induction of pro-angiogenic Gal1 in hypoxic tumormicroenvironments. Supporting these findings, Gal1 preferentiallylocalized within hypoxic regions surrounding necrotic areas in thecenter of KS xenografts (FIG. 18I).

Having defined the molecular pathways underlying hypoxia-regulated ECcell surface glycosylation and tumor Gal1 expression, it was nextdetermined whether Gal1-glycan lattices could couple tumor hypoxia toangiogenesis at the tumor-EC interface. To address this questiondirectly, a series of in vitro and in vivo experiments were performed todisrupt lattice formation either by blocking Gal1 expression orhindering N- or O-glycan elongation. Three different short hairpin RNAconstructs targeting unique sequences of Gal1 (shGal1.1, shGal1.2,shGal1.3) were stably expressed in KS cells. Retroviral-mediatedinfection of KS cells with shGal1.1 or shGal1.2 suppressed Gal1expression substantially under both normoxic and hypoxic conditions(FIG. 18S). Serum-free conditioned medium (CM) obtained from KS cellsexposed to hypoxic conditions induced a 3-fold increase in the formationof EC tubular networks compared to KS cells incubated under normoxicconditions; this effect was eliminated when Gal1, VEGF or both wereknocked down in KS cells (FIG. 18J). Additionally, CM from KS cellscultured in hypoxic microenvironments augmented angiogenesis whenincorporated in vivo into Matrigel plugs (FIG. 18K). However, hypoxic KSCM failed to induce angiogenesis when cells were stably transfected withGal1 shRNA. This effect proceeded irrespectively of whether CM from Gal1knockdown KS clones were implanted into wild-type or Gal1-deficient(Lgals1^(−/−)) mice (FIG. 18K), suggesting that hypoxia-regulated,tumor-derived Gal1 contributes to angiogenesis independently of thepresence or absence of the host endogenous lectin. To substantiatefurther the relevance of Gal1-glycan lattices as bridging partners ofhypoxia-driven angiogenesis, CM from hypoxic KS cells with ECstransfected with GnT5 or GCNT1 siRNA was assayed. Interruption ofcomplex N-glycan branching prevented full induction of tubular networksstimulated by hypoxic KS cells, whereas hampering core-2 O-glycanelongation had no effect (FIG. 18L), underscoring the critical role ofcomplex N-glycans in coupling tumor hypoxia to HIF-1α-independentangiogenesis.

Given the critical role of VEGF in angiogenesis and vasculogenesis andthe co-option of Gal1 for VEGFR2 signaling, the reciprocal regulation ofthese pro-angiogenic mediators by establishing single or doubleknockdown KS clones was further analyzed. No substantial differencescould be detected in the magnitude of VEGF or Gal1 secretion amongwild-type, Gal1 knockdown or VEGF knockdown KS cells incubated undernormoxic or hypoxic conditions (FIGS. 18T and 18U), indicating lack ofcross-regulation between these pro-angiogenic mediators. Thus,lectin-glycan lattices can form signaling clusters that bridge tumorhypoxia to angiogenesis through mechanisms that are dependent of ROS andNF-κB but are independent of HIF-1α and VEGF.

Example 11 Targeted Disruption of Galectin-1-Glycan Lattices In VivoPrevents Tumor Angiogenic Switch

To delineate the pathophysiologic role of Gal1-glycan lattices, theconsequences of Gal1 inhibition in a xenograft model of human KS in nudemice were assessed, which enables the examination of Gal1 function intumor vascularization separately from its role in T cell-dependentimmunity. Human knockdown KS clones expressing Gal1 shRNA, control KScells expressing scrambled shRNA (sh-scr) or wild-type KS cells (FIG.18S) were implanted into the flanks of nude mice. Inoculation of Gal1knockdown KS clones led to a considerable reduction in tumor growth(sh-Gal1.1: 51.2%; sh-Gal1.2: 60.6% at day 22 post-inoculation) comparedto mice receiving control KS cells (FIG. 19A). This effect was not dueto intrinsic differences in proliferation rates, as control KS cellsshowed no growth advantage in vitro over Gal1 knockdown clones (FIG.19G).

Gal1 silencing also attenuated the formation of a typical high densitymicrovessel network, as reflected by a substantial decline in the levelsof tumor hemoglobin content and the percentage of CD34⁺ ECs (FIGS. 19B,19C, and 19H). These results were verified using antisense RNAstrategies (FIGS. 19I-19L). Gal1 transcript was part of the human andmouse KS molecular signature as Gal1 was overexpressed in human AIDS-KS,as well as in mECK36, a murine model of KSHV-induced KS tumors (Mutlu etal. (2007) Cancer Cell 11, 245-258) (FIGS. 19D, 19E, and 19M). Moreover,in patient biopsies, Gal1 was selectively expressed in KS lesionsassociated with vascular channels, showing robust cytoplasmic and weakmembrane staining in spindle cells. In contrast, Gal1 was barelydetected in all benign vascular lesions analyzed, includingtelangiectatic hemangioma, benign lymphangioendothelioma and pyogenicgranuloma, in which only diffuse staining of the inflammatoryinfiltrates was detected (FIGS. 19F and 19N), indicating an additionalrole for Gal1 as a diagnostic biomarker capable of delineating highlyangiogenic human KS from benign vascular lesions with shared morphologicand molecular features.

In addition, an emerging area in cancer therapy involves theidentification of multi-targeted agents capable of concurrently shapingvascular and immune compartments (Jinushi et al. (2007) Clin Cancer Res13, 3762-3764). In order to integrate the individual roles ofGal1-glycan lattices (i.e., remodeling vascular networks and dampening Tcell immunity) and to assess directly the dual benefits of targetingthese interactions, the effects of Gal1 inhibition in the B16 melanomamodel in immunocompetent hosts was studied. Syngeneic mice inoculatedwith knockdown B16 clones expressing shRNA constructs (FIGS. 20A and20B) showed diminished tumor burden and reduced number oftumor-associated ECs compared to mice injected with melanoma cellsexpressing control shRNA (FIGS. 20A-20C). Tumor-draining lymph node(TDLN) cells from mice receiving knockdown clones had increasedproliferation and greater secretion of IFN-γ and IL-17 after ex vivorestimulation with B16 cells (FIGS. 20D and 20E) and showed a markeddecline in the frequency of CD4 CD25 FoxP3 T regulatory (T_(reg)) cells(FIG. 20F), as compared to lymph node cells from mice receiving controltransfectants. This effect was slightly but significantly morepronounced when shRNA B16 clones were inoculated into syngeneicLgals1^(−/−) mice, indicating a modest contribution of host-derived Gal1to this effect. These results indicate that interruption of Gal1-glycanlattices may serve to limit tumor growth by simultaneously targetingimmune and vascular compartments.

Given the extensive remodeling of EC surface glycans imprinted byproliferative, tolerogenic and hypoxic stimuli (FIGS. 16B and 18A), itwas hypothesized that changes in glycosylation may selectively occur invivo in tumor-associated versus normal vasculature. When compared toblood vessels within normal skin, tumor-associated vasculature displayedhigher frequency of L-PHA-reactive glyco-epitopes and lower SNAreactivity, indicating increased β1-6 N-glycan branching and decreasedα2-6-linked SA (FIG. 20G). Thus, differential glycosylation oftumor-associated versus normal vasculature may facilitate Gal1signaling, lattice formation and promotion of angiogenic switch.Furthermore, profiling of a series of human primary melanomasestablished a highly significant positive correlation between tumorexpression of Gal1 and microvessel density (FIG. 20H), supporting theclinical relevance of the Gal1-glycan axis in tumor vascularity and itstherapeutic value in human cancer settings.

Example 12 Therapeutic Administration of a Galectin-1-SpecificNeutralizing mAb Promotes Vascular Remodeling and Influx of ImmuneEffector Cells

Having established the benefits of disrupting Gal1-glycan lattices intumor microenvironments, the effects of a recently developedneutralizing Gal1 monoclonal antibody (mAb), 8F4F8G7 (Example 1), whichprevented the binding of Gal1 to human ECs, were determined (FIGS. 21Aand 21I). This mAb was specific for Gal1 since it did not interfere withthe binding of other members of the galectin family, such as Gal3 orGal8, to the EC surface (FIG. 21J). The functional activity of this mAbwas demonstrated in vitro through its specific capacity to prevent ECproliferation, migration and capillary tube formation induced by Gal1,but not VEGF (FIGS. 21B-21D). Notably, the 8F4F8G7 mAb did not alter ECbiology in the absence of exogenous Gal1 (FIGS. 21C and 21D). Moreover,8F4F8G7 mAb specifically inhibited VEGFR2 phosphorylation in response toGal1 to levels comparable to those observed by GnT5 silencing (FIG.21E); this finding further substantiates a key role for VEGFR2 inmediating Gal1 signaling, lattice formation and angiogenic sprouting.

To validate the therapeutic potential of interrupting Gal1 signaling invivo, different doses of the 8F4F8G7 mAb (2.5 mg/kg, 7.5 mg/kg or 15mg/kg) or the isotype control were infused into nude mice bearingestablished KS tumors. Treatment of nude mice with 8F4F8G7 mAb induced adose-dependent delay in tumor growth (FIGS. 21F and 21K). Moreover,administration of 8F4F8G7 mAb, but not its isotype control, afforded asignificant reduction in tumor microvasculature (FIGS. 21G and 21H),indicating that mAb-mediated Gal1 blockade attenuates aberrantneovascularization.

To analyze vascular and immune compartments simultaneously, thetherapeutic value of 8F4F8G7 mAb in the syngeneic B16 model, in whichmicrovessel networks are more clearly distinguishable from the tumorparenchyma, was determined. Administration of the anti-Gal1 mAb toimmunocompetent mice bearing established B16 tumors resulted in markedlydecreased tumor burden (−86% at day 20), while injection intoimmunodeficient B6/Rag^(−/−) mice showed only a partial anti-tumoreffect (FIGS. 22A and 22M). No significant changes in the frequency ofCD34 cells at day 20 (FIG. 22N) were detected and only a slight decreasein microvessel density of tumors obtained 30 days post-inoculation wasfound. However, interruption of Gal1 signaling through mAb-mediatedblockade resulted in substantial remodeling of tumor vasculature (FIGS.22B-22E). While B16 tumors treated with an isotype control mAb displayeda chaotic and heterogeneous vascular architecture composed of extensivesprouting and large vessels fused to microvessels, the resultant tumorvasculature of mice treated with the 8F4F8G7 mAb resembled normalvascular networks with regard to vessel diameter and distribution (FIGS.22B and 22C). The resultant vasculature in 8F4F8G7 mAb-treated miceincluded fewer dilated and tortuous vessels (FIGS. 22B and 22C) andgreater coverage by pericytes (FIG. 22D). Most pericytes in 8F4F8G7mAb-treated tumors displayed a more mature phenotype, as revealed byhigher expression of α-smooth muscle actin (αSMA) and lower expressionof regulator of G-protein signaling 5 (Rgs5) and platelet-derived growthfactor receptor (PDGFR)β, when compared to pericytes fromisotype-treated tumors (FIGS. 22D, 22E, and 22O). Yet, no significantvariations were detected in the expression of desmin between8F4F8G7-treated and isotype-treated tumors (FIG. 22E). These phenotypicchanges typically delineate the transition from an immature to a maturepericyte profile (Hamzah et al. (2008) Nature 453, 410-414). Supportingthese results, administration of 8F4F8G7 mAb, but not its isotypecontrol, markedly alleviated tumor hypoxia as shown by reduced formationof pimonidazole adducts (FIG. 22F). Thus, blockade of Gal1 signalingcounteracts the aberrant nature of tumor vasculature not only byattenuating vessel sprouting but also by modulating vascular morphologyor influencing pericyte coverage and maturation early during treatment.

Given the lack of a single therapeutic agent capable of simultaneouslytargeting vascular and immune compartments, it was further investigatedwhether vascular remodeling induced by 8F4F8G7 mAb was accompanied byaugmented anti-tumor immune response. Therapeutic administration of the8F4F8G7 mAb stimulated proliferation as well as the synthesis andsecretion of IFN-γ and IL-17 by tumor-draining lymph node cellsrestimulated ex vivo with B16 cells (FIGS. 22G, 22H, and 22P). Incontrast, interruption of Gal1 signaling blunted B16-specific IL-10production (FIG. 22O). This cytokine profile, reflecting unleashedeffector responses, was further supported by a decline in the frequencyof CD4⁺CD25⁺FoxP3⁺T_(reg) cells in TDLN cells from mice receiving8F4F8G7 mAb versus those given isotype control (FIGS. 22I and 22Q).Moreover, a dramatic increase in the number of tumor-infiltratingIFN-γ-producing CD8⁺ T cells was detected in 8F4F8G7 mAb-versusisotype-treated mice (FIGS. 22J and 22R).

To evaluate whether the augmented immune response was, at least in part,mediated by the increased influx of immune cells due to vesselremodeling, T cells obtained from mice harboring B16 tumors were labeledwith the CFSE dye and adoptively transferred into tumor-bearingrecipient mice treated with 8F4F8G7 mAb or isotype control. A greaternumber of T cells reached tumor parenchyma (3-fold increase) in micereceiving 8F4F8G7 mAb, as compared to those treated with control isotype(FIGS. 22K and 22R), indicating enhanced influx of immune cellssubsequent to vessel remodeling. In contrast, there were no differencesin the number of CFSE⁺ T cells in spleens of recipient mice (FIG. 22K).To rule out the possibility that Gal1 blockade affects immune cellrecruitment by influencing chemotaxis rather than vascular remodeling,similar experiments were performed using fluorescently-labeled beads asa non-cellular approach. In vivo tracking revealed increased access offluorescently-labeled beads to the tumor parenchyma of 8F4F8G7mAb-treated as compared to isotype-treated mice (FIG. 22L).

Taken together, these results identify the first Gal1-specific agentcapable of affording therapeutic benefits by attenuating abnormalangiogenesis and facilitating vascular remodeling and influx of immuneeffector cells, which, in the absence of Gal1 signaling, are morecompetent for limiting tumor growth.

Lectin-glycan lattices are spatial arrays of glycans and endogenousmultivalent lectins that organisms use to decode the biologicalinformation present in their own ‘glycome’ (Paulson et al. (2006) NatChem Biol 2, 238-248). Recent efforts toward deciphering thisinformation revealed dramatic changes in the repertoire of N- andO-glycans in the transition from normal to inflamed or neoplastictissue, providing novel opportunities for differential diagnosis andtherapeutic intervention (Dube et al. (2005) Nat Rev Drug Discov 4,477-488). The results described herein describe a vascular circuit,regulated by lectin-glycan lattices, which couples tumor hypoxia toabnormal neovascularization. The results demonstrate that hypoxic,proliferative, tolerogenic or inflammatory stimuli differentiallyregulate the glycosylation signature of ECs, allowing or preventing theformation of Gal1-glycan lattices. These signaling clusters cansubstitute for canonical ligands such as VEGF, modulate EC biology andpreserve the angiogenic phenotype. Tumor hypoxia selectively amplifiesthis circuit by shaping the repertoire of N-glycans on VEGFR2 andaugmenting Gal1 synthesis through mechanisms involving ROS-mediatedNF-κB activation. Targeting Gal1-glycan lattices in vivo limits tumorgrowth by attenuating hypoxia-driven angiogenesis and favoringremodeling of tumor vascular networks, as shown by increased pericytecoverage and maturation, alleviation of tumor hypoxia and increasedinflux and expansion of tumor-specific immune cells.

The results described herein further demonstrate that Gal1-specificneutralizing mAbs attenuate tumor angiogenesis and promotes vascularremodeling by increasing pericyte coverage and maturation.Antibody-mediated Gal-1 blockade alleviates tumor hypoxia and fostersthe influx of anti-tumor immune cells into the tumor bed. This vascularremodeling function recapitulates that observed with otheranti-angiogenic agents, which can transiently normalize tumorvasculature to make it more efficient for oxygenation, drug delivery,and immune cell entry (Jain, R. K. (2005) Science 307, 58-62). Moreover,as bone marrow-derived myeloid cells express considerable amounts ofGal1 (Ilarregui et al. (2009) Nat Immunol 10, 981-991), its inhibitionmight also contribute to eliminate the vasculogenic potential of thesecells. Although galectin inhibitors that block the carbohydraterecognition domain have been developed (Ingrassia et al. (2006) Curr MedChem 13, 3513-3527; Stannard et al. (2010) Cancer Lett. [Epub ahead ofprint]), most of these inhibitors lack selectivity for individualmembers of the galectin family and often display weak ligand affinitiesand poor bioavailability. These shortcomings hinder the rapidtranslation of these compounds into the clinic, underscoring theadvantages of a mAb that specifically neutralizes Gal1 and targets bothvascular and immune compartments.

Moreover, the results described herein demonstrate a strong correlationbetween Gal1 expression and the extent of tumor angiogenesis in humanmelanoma biopsies. In addition, Gal1 expression delineated highlyangiogenic KS from benign vascular lesions with shared morphologic andmolecular features, indicating its potential use as a differentialdiagnostic biomarker in vascular malignancies. These data haveadditional implications as Gal1 blockade may ameliorate AIDS-related KSnot only by limiting aberrant angiogenesis, but also by restoring thebalance between T_(H)17 and T_(reg) cell populations (Favre et al,2009).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide web attigr.org and/or the National Center for Biotechnology Information (NCBI)on the world wide web at ncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the present invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. A monoclonal antibody, or antigen-bindingfragment thereof, that specifically binds galectin1 (Gal1) comprisingsix CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, whereinCDR-H1 consists of residues 31-35 of SEQ ID NO: 7, CDR-H2 consists ofresidues 50-66 of SEQ ID NO: 7, CDR-H3 consists of residues 99-107 ofSEQ ID NO: 7, CDR-L1 consists of residues 23-36 of SEQ ID NO: 9, CDR-L2consists of residues 52-58 of SEQ ID NO: 9, and CDR-L3 consists ofresidues 91-99 of SEQ ID NO:
 9. 2. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, comprising the heavy chainvariable domain sequence of SEQ ID NO:
 7. 3. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, comprising the light chainvariable domain sequence of SEQ ID NO:
 9. 4. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, comprising the heavy chainvariable domain sequence of SEQ ID NO: 7 and the light chain variabledomain sequence of SEQ ID NO:
 9. 5. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, wherein the monoclonalantibody, or antigen-binding fragment thereof neutralizes Gal1.
 6. Themonoclonal antibody, or antigen-binding fragment thereof, of claim 5,wherein the monoclonal antibody, or antigen-binding fragment thereofneutralizes human Gal1, mouse Gal1, or cynomologous monkey Gal1.
 7. Themonoclonal antibody, or antigen-binding fragment thereof, of claim 1,wherein the monoclonal antibody, or antigen-binding fragment thereofblocks the interaction between Gal1 and VEGFR2.
 8. The monoclonalantibody, or antigen-binding fragment thereof, of claim 1, wherein themonoclonal antibody, or antigen-binding fragment thereof inhibitsGal1-induced T cell apoptosis, stimulates IFN-γ production of lymph nodecells in the presence of melanoma cells, stimulates IL-17 production oflymph node cells in the presence of melanoma cells, stimulatesintratumoral CD8+ T cell influx in a melanoma tumor, inhibitsendothelial cell proliferation induced by Gal1, inhibits endothelialcell migration induced by Gal1, inhibits capillary tube formationinduced by Gal1, inhibits melanoma tumor hypoxia, inhibits angiogenesis,increases blood vessel pericyte coverage, or increases pericytematuration.
 9. The monoclonal antibody, or antigen-binding fragmentthereof, of claim 1, wherein the monoclonal antibody, or antigen-bindingfragment thereof, is a humanized antibody, a chimeric antibody, a Fabfragment, a F(ab′)₂ fragment, or an Fv fragment.
 10. The monoclonalantibody, or antigen-binding fragment thereof, of claim 1, wherein themonoclonal antibody, or antigen-binding fragment thereof, comprises animmunoglobulin heavy chain constant domain selected from the groupconsisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG3, IgG4, IgA, IgM, andIgE constant domains.
 11. The monoclonal antibody, or antigen-bindingfragment thereof, of claim 1, wherein the monoclonal antibody, orantigen-binding fragment thereof, is conjugated to an agent selectedfrom the group consisting of a cytotoxic agent, a drug, an enzyme, aprosthetic group, a fluorescent material, a luminescent material, abioluminescent material, and a radioactive material.
 12. A device or kitcomprising at least one monoclonal antibody, or antigen-binding fragmentthereof, of claim 1, said device or kit optionally comprising a label todetect the at least one monoclonal antibody or antigen-binding fragmentthereof, or a complex comprising the monoclonal antibody orantigen-binding fragment thereof.
 13. A pharmaceutical compositioncomprising the antibody, or antigen-binding fragment thereof, of claim1, in a pharmaceutically acceptable carrier.
 14. The hybridoma 14-198F4-F8-G7 deposited under accession number PTA-10535.